Compositions and Methods for Treating Liquid Cancers

Abstract

As described below, the present invention provides genetically modified immune cells, said immune cells having enhanced anti-tumor formation activity, anti-immunosuppression, and a reduced risk of causing graft-versus-host reaction, or a composition thereof. The present invention also provides methods for producing and using these modified immune effector cells.

Description

[0001] Cross - reference to related applications

[0002] This application is an international PCT application that claims the priority and benefit of U.S. Provisional Patent Application 62 / 907,254, filed on September 27, 2019, the entire content of which is incorporated herein by reference in its entirety. Technical field

[0003] This application relates to genetically modified immune cells having enhanced anti - tumorigenic activity, anti - immunosuppression, reduced risk of causing graft - versus - host reaction, or a combination thereof. This application also relates to methods for producing and using such genetically modified immune cells. Background art

[0004] Autologous and allogeneic immunotherapies are cancer treatment methods in which immune cells expressing chimeric antigen receptors are administered to a subject. To generate immune cells expressing chimeric antigen receptors (CARs), immune cells are first collected from a subject (autologous) or a donor separate from the subject being treated (allogeneic) and genetically modified to express a chimeric antigen receptor. The resulting cells express the 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 tumor cells. This interaction with the tumor marker activates the CAR - T cells, which then kill the tumor cells. However, for autologous or allogeneic cell therapies to be effective or efficient, significant conditions and cellular responses such as T - cell signaling inhibition must be reduced or avoided. For allogeneic cell therapies, graft - versus - host disease and host rejection of CAR - T cells can pose additional challenges. Editing the genes involved in these processes can enhance CAR - T cell function and resistance to immune repression or inhibition, when current methods used to make such edits have the potential to induce large genomic rearrangements in CAR - T cells, thereby negatively impacting their efficacy. Accordingly, there is a significant need for techniques to more precisely modify immune cells, especially CAR - T cells. This application addresses this and other important needs. Summary of the invention

[0005] As described below, the present invention features genetically modified immune cells that have enhanced anti-tumorigenic activity, resistance to immune suppression, and a reduced risk of causing graft-versus-host disease or host-versus-graft reaction, where host CD8+ T cells recognize the graft as non-self (e.g., where the graft recipient generates an immune response against the transplanted organ), or a combination thereof. In one embodiment, CAR-T cells lacking or having reduced levels of functional TRAC are administered to a subject having graft-versus-host disease (GVHD) or having a predisposition to develop GVHD. In one embodiment, CAR-T cells lacking or having reduced levels of β2-microglobulin (B2M) are administered to a subject having host-versus-graft disease (HVGD) or having a predisposition to develop HVGD. The present invention also provides methods for producing and using these modified immune cells.

[0006] On the one hand, the present invention provides a composition comprising two or more immune cells, each immune cell comprising a) a different chimeric antigen receptor that targets 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 the expression of the targeted antigen; and b) one or more mutations that reduce or eliminate the 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 that targets CD5, and another immune cell comprises a chimeric antigen receptor that targets 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 that targets 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 that targets CD7, and another immune cell expresses a chimeric antigen receptor that targets 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 that targets CD3, and another immune cell expresses a chimeric antigen receptor that targets an antigen selected from the group consisting of CD33 and CD123. In some embodiments, one of the immune cells expresses a chimeric antigen receptor that targets CD33, and another immune cell expresses a chimeric antigen receptor that targets 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 another 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 another 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 another 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 another chimeric antigen receptor targets CD123.

[0007] On the other hand, the present invention 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 the expression of the targeted antigen; and further comprise one or more mutations that reduce or eliminate the expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0008] In yet another aspect, the present invention 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 the expression of the targeted antigen; and further comprise one or more mutations that reduce or eliminate the expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0009] In one aspect, the present invention provides a composition comprising at least two immune cells, each comprising a different chimeric antigen receptor, wherein one chimeric antigen receptor targets CD5, and the other targets CD7, wherein the cells further comprise a mutation that reduces or eliminates the expression of the targeted antigen; and further comprise one or more mutations that reduce or eliminate the expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0010] On the other hand, the present invention 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 CD5, wherein the cells further comprise a mutation that reduces or eliminates the expression of the targeted antigen; and further comprise one or more mutations that reduce or eliminate the 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 present invention 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 the expression of the targeted antigen; and further comprises one or more mutations that reduce or eliminate the expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0012] 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 the expression of the encoded 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 the expression of the targeted antigen and / or the immunogenic polypeptide. In some embodiments, the immune cells are fratricide-resistant. In some embodiments, the immune cells have increased anti-tumorigenic activity. In some embodiments, the immune cells do not comprise a detectable translocation. 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 progenitor is a hematopoietic stem cell.

[0013] In one aspect, the present invention provides a pharmaceutical composition comprising an effective amount of any composition provided herein and a pharmaceutically acceptable excipient.

[0014] On the other hand, the present invention provides a base editor system comprising a fusion protein, the fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase domain, and at least two guide polynucleotides, each guide polynucleotide 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 another targets a member selected from the group consisting of CD7, CD3, CD33, and CD123. In some embodiments, the system comprises three guide polynucleotides, each guide polynucleotide targeting one of the antigens CD3, CD5, and CD7. In some embodiments, one guide polynucleotide targets CD7 and another targets a member selected from the group consisting of CD3, CD33, and CD123. In some embodiments, one guide polynucleotide targets CD3 and another targets a member selected from the group consisting of CD33 and CD123. In some embodiments, one guide polynucleotide targets CD33 and another targets CD123.

[0015] In some embodiments, the guide polynucleotides each comprise a nucleic acid sequence selected from Table 26. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from AGCGACUGCAGAAAGAAGAG or CAUACCAGCUGAGCCGUCCG. In some embodiments, the fusion protein further comprises one or more uracil glycosylase inhibitors (UGI) and / or one or more nuclear localization sequences (NLS). In some embodiments, the napDNAbp comprises a Cas9, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, or Cas12j / CasΦ polypeptide or a portion thereof. In some embodiments, the napDNAbp comprises a Cas12 polypeptide or a fragment thereof. In some embodiments, the napDNAbp comprises a Cas9 polypeptide or a fragment thereof. In some embodiments, the Cas9 is dead Cas9 (dCas9) or Cas9 nickase (nCas9). In some embodiments, the Cas9 is modified Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), or modified Streptococcus pyogenes Cas9 (SpCas9). In some embodiments, the Cas9 comprises an altered protospacer adjacent motif (PAM) specificity. In some embodiments, the altered PAM is specific for the nucleic acid sequence 5'-NGC-3'.

[0016] 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.

[0017] In one aspect, the present invention provides a pharmaceutical composition comprising any of the base editor systems provided herein.

[0018] In another aspect, the present invention provides a polynucleotide encoding any of the base editor systems and guide polynucleotides provided herein.

[0019] In yet another aspect, the present invention 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 herpesviral vector, or an adeno-associated viral vector (AAV).

[0020] In one aspect, the present invention provides a pharmaceutical composition comprising any of the polynucleotides or any of the vectors provided herein.

[0021] On the one hand, the present invention provides a method for producing CAR-expressing immune cells with reduced immunogenicity. In some embodiments, the method comprises expressing a base editor system in the CAR-expressing immune cells, the base editor system comprising a fusion protein and two guide polynucleotides, the fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase domain, each of the guide polynucleotides 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 CAR-expressing immune cells with reduced immunogenicity. In some embodiments, the immune cells express or are contacted with guide polynucleotides that target polynucleotides encoding polypeptides selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0022] In some embodiments, the method comprises (a) expressing a base editor system in the CAR-expressing immune cells, the 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 cells with at least two guide polynucleotides, each of the guide polynucleotides 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 CAR-expressing immune cells with reduced immunogenicity.

[0023] In some embodiments, the method further comprises contacting the CAR-expressing immune cells with guide polynucleotides that target polynucleotides encoding polypeptides selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRB. In some embodiments, the immune cells express a chimeric antigen receptor (CAR) that targets 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 cells that reduces or eliminates the 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 with reduced immunogenicity.

[0024] On the one hand, the present invention provides a method for producing a population of CAR-expressing immune cells having reduced immunogenicity. In some embodiments, the method comprises a) introducing into an immune cell a mutation that reduces or eliminates the expression of an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123 and introducing a different mutation in one of said antigens into a second immune cell; and b) introducing into the immune cell a mutation that reduces or eliminates the 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 cells produced by the method express a chimeric antigen receptor targeting the CD3, CD5, and / or CD7 antigen but fail to express or express at reduced levels the CD3, CD5, and / or CD7 antigen. In some embodiments, the immune cells produced by the method express a chimeric antigen receptor targeting the CD33 and CD123 antigen but fail to express or express at reduced levels 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 the CD5 CAR construct presented in Table 28.

[0025] On the other hand, the present invention provides a method for producing immune cells having reduced immunogenicity. In some embodiments, the method comprises a) introducing into the endogenous CD5 gene sequence or regulatory element a mutation that reduces or eliminates the expression of CD5; and b) expressing in said cell the CD5 CAR construct presented in Table 28. In some embodiments, the CD5 CAR construct encodes a CD5 CAR polypeptide comprising or consisting of an amino acid sequence selected from:

[0026] a)

[0027]

[0028]

[0029] b)

[0030]

[0031] c)

[0032]

[0033] d)

[0034]

[0035]

[0036] e)

[0037]

[0038] In some embodiments, the immune cells produced by the method exhibit fratricide resistance and / or increased antitumorigenic activity compared to corresponding control cells. In some embodiments, the method is performed in vivo or ex vivo. In some embodiments, the immune cells generated by the method do not contain detectable translocations. In some embodiments, the immune cells generated by the method contain less than 1% indels. In some embodiments, the immune cells generated by the method contain less than 5% off-target editing. In some embodiments, the immune cells generated by the method contain less than 5% non-target editing. In some embodiments, 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, one or more mutations are generated by contacting a 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.

[0039] In some embodiments, the deaminase is adenosine or cytidine deaminase. In some embodiments, the cytidine deaminase is BE4. In some embodiments, the mutation reduces the expression of the encoded 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, one or more nucleic acid sequences each target napDNAbp to the 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 immune cells by electroporation, nucleofection, cationic lipid-mediated methods, viral transduction, or a combination thereof. In some embodiments, the method further comprises expanding the immune cells in culture to generate a population of immune cells. In some embodiments, the 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 TCRα / β+ cells from the modified population of immune cells.

[0040] In one aspect, the invention provides CAR-expressing immune cells having reduced immunogenicity produced by any of the methods provided herein.

[0041] In another aspect, the invention provides a pharmaceutical composition comprising any of the immune cells provided herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0042] In yet another aspect, the invention provides a method for killing tumor cells. In some embodiments, the method comprises contacting tumor cells expressing an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123 with two or more immune cells, each expressing a different chimeric antigen receptor that targets two of the antigens expressed by the cell, wherein the immune cells comprise one or more mutations that reduce or eliminate the expression of the targeted antigen, and one or more mutations that reduce or eliminate the 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 cells are derived from tumor formation.

[0043] On the one hand, the present invention provides a method for treating tumorigenesis 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 that targets an antigen expressed by the tumor cells of the subject, the antigen being 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 the expression of the antigen, and each of the immune cells further comprises one or more mutations that reduce or eliminate the expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0044] In some embodiments, the tumorigenesis is a hematological cancer. In some embodiments, the tumorigenesis is a liquid cancer. In some embodiments, the hematological cancer is leukemia, myeloma, and / or lymphoma. In some embodiments, the hematological cancer is a B-cell cancer. In some embodiments, the hematological cancer is selected from at least one of the following: 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 leukemia, angioimmunoblastic T / NK-cell lymphoma, hepatosplenic T-cell lymphoma, primary cutaneous CD30 + lymphoproliferative disorder, extranodal NK / T-cell lymphoma, adult T-cell leukemia / lymphoma, T-cell prolymphocytic leukemia, subcutaneous panniculitis-like T-cell lymphoma, primary cutaneous gamma / delta T-cell lymphoma, aggressive NK-cell leukemia, and enteropathy-associated T-cell lymphoma. In some embodiments, the hematological cancer is T-cell acute lymphoblastic leukemia (T-ALL) cells. In some embodiments, the hematological cancer is T-cell acute myeloid leukemia (AML).

[0045] On the other hand, the present invention provides a method for treating tumorigenesis 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 that targets an antigen expressed by the subject's tumor cells, 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 the expression of the antigen, and each of the immune cells further comprises one or more mutations that reduce or eliminate the 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 tumorigenesis expressing an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123. In some embodiments, the hematological cancer is T cell acute lymphoblastic leukemia (T-ALL) cells. In some embodiments, the hematological cancer is 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.

[0046] In yet another aspect, the present invention provides a method for antigen-dependently killing a subject's tumor cells. In some embodiments, the method comprises administering to a subject having tumorigenesis 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 that targets one of the antigens, wherein the immune cells comprise one or more mutations that reduce or eliminate the expression of the targeted antigen, and one or more mutations that reduce or eliminate the expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0047] In one aspect, the present invention provides a method for antigen-dependently killing a subject's acute myeloid leukemia (AML) cells. In some embodiments, the method comprises administering to a subject having AML expressing CD33 and CD123 antigens two or more immune cells, each expressing a different chimeric antigen receptor that targets one of the antigens, wherein the immune cells comprise one or more mutations that reduce or eliminate the expression of the targeted antigen, and one or more mutations that reduce or eliminate the expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0048] On the one hand, the present invention provides a method for antigen - dependently killing T - cell acute lymphoblastic leukemia (T - ALL) cells of 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 that targets one of the antigens, wherein the immune cells comprise one or more mutations that reduce or eliminate the expression of the targeted antigen, and one or more mutations that reduce or eliminate the expression of a polypeptide selected from the group consisting of TRAC, LAG - 3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0049] On the other hand, the present invention provides a method for treating 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 that targets the CD33 or CD123 antigen, wherein the immune cells comprise one or more mutations that reduce or eliminate the expression of the targeted antigen, and one or more mutations that reduce or eliminate the 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.

[0050] In yet another aspect, the present invention provides a method for treating 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 that targets the CD3, CD5, and CD7 antigens, wherein the immune cells comprise one or more mutations that reduce or eliminate the expression of the targeted antigen, and one or more mutations that reduce or eliminate the 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.

[0051] In some embodiments, the immune cells are cytotoxic T cells, regulatory T cells, T helper cells, dendritic cells, B cells, or NK cells. In some embodiments, the subject has been previously treated with lymphodepletion. In some embodiments, 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.

[0052] In one aspect, the invention provides a kit comprising any of the compositions provided herein for treating cancer. In another aspect, the invention provides a kit comprising any of the base editor systems provided herein for generating CAR-expressing immune cells with reduced immunogenicity. In some embodiments, any of the kits provided herein comprises written instructions for using the kit.

[0053] The descriptions and examples herein illustrate embodiments of the disclosure in detail. It should be understood that the disclosure is not limited to the specific embodiments described herein and may thus vary. Those skilled in the art will recognize that there are numerous variations and modifications of the disclosure that are encompassed within the scope of the disclosure.

[0054] Unless otherwise indicated, the practice of some embodiments disclosed herein employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genetics, and recombinant DNA, which are within the capabilities of the art. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, Fourth Edition (2012); Current Protocols in Molecular Biology series (F.M. Ausubel, et al. eds.); Methods In Enzymology series (Academic Press, Inc.); PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. 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, Sixth Edition (R.I. Freshney, ed. (2010).

[0055] Although the various features of the disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any combination. Conversely, although the invention may be described in the context of separate embodiments for clarity, the invention may also be implemented in a single embodiment. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0056] The features of the disclosure are set forth in particularity in the appended claims. A better understanding of the features and advantages of the disclosure can be obtained by reference to the following detailed description of exemplary embodiments that make use of the principles of the disclosure, and to the drawings described below.

[0057] Definitions

[0058] The following definitions supplement those of the art and are specific to this application and are not applicable to any related or unrelated case, e.g., any reissued patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred materials and methods are as described herein. Accordingly, the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0059] Unless otherwise defined, all technical terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Dictionary of Microbiology and Molecular Biology (2nd ed. 1994) (Singleton et al.); 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).

[0060] In this application, unless specifically stated otherwise, the use of the singular includes the plural. It must be noted that, as used in this specification, unless the context clearly indicates otherwise, the singular forms "a" and "the" include plural referents. In this application, unless otherwise specified, the use of "or" means "and / or" and is understood to be inclusive. In addition, the use of the term "comprising" and other forms such as verb forms, active or passive forms, is unrestricted.

[0061] As used in this specification and the claims, the words "comprising" (and any form of comprising, such as singular and plural forms), "having" (and any form of having, such as singular and plural forms), "including" (and any form of including, such as singular and plural forms), or "containing" (and any form of containing, such as singular and plural forms) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0062] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by a person of ordinary skill in the art, and will depend in part on how the value is measured or determined, i.e., the limitations of the measuring system. For example, "about" can mean within one (1) or more standard deviations, according to the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly with respect to biological systems or lengths, the term can mean within an order of magnitude, such as within 5-fold or 2-fold of a value. When a particular value is described in the specification and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0063] The ranges provided herein are understood to be shorthand for all values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 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.

[0064] "Some embodiments", "embodiments", "one embodiment", or "other embodiments" in the specification mean that the particular features, structures, or characteristics described with respect to the embodiments are included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0065] "Adenosine deaminase" means a polypeptide or a fragment thereof 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, 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 bacteria.

[0066] 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 Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Salmonella typhi (S. typhi), Shewanella putrefaciens (S. putrefaciens), Haemophilus influenzae (H. influenzae), Caulobacter crescentus (C. crescentus), or Geobacter sulfurreducens (G. sulfurreducens). In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is an Escherichia coli TadA (ecTadA) deaminase or a fragment thereof.

[0067] In some embodiments, the ecTadA cytidine deaminase is ecTadA. For example, truncated ecTadA may lack one or more N-terminal amino acids relative to full-length ecTadA. In some embodiments, relative to full-length ecTadA, 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. In some embodiments, relative to full-length ecTadA, 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. In some embodiments, the ecTadA deaminase does not contain an N-terminal methionine. In some embodiments, the TadA deaminase is an N-terminally truncated TadA. In certain embodiments, TadA is any of the TadA's described in PCT / US2017 / 045381, which is incorporated herein by reference.

[0068] 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.

[0069] "Adenosine deaminase base editor 8 (ABE8) polypeptide" or "ABE8" means a base editor as defined herein that includes an adenosine deaminase variant that includes alterations at amino acid positions 82 and / or 166 of the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0070] In some embodiments, ABE8 includes further alterations as described herein relative to the reference sequence.

[0071] "Adenosine deaminase base editor 8 (ABE8) polypeptide" means a polynucleotide encoding ABE8.

[0072] "Adenosine deaminase base editor 9 (ABE9) polypeptide" or "ABE9" means a base editor containing an adenosine deaminase variant as defined herein, the adenosine deaminase variant containing 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, which alterations are in the following reference sequences:

[0073]

[0074] The relevant bases altered in the reference sequences are shown in underlined and bold font. In some embodiments, ABE9 contains further alterations as described herein relative to the reference sequences. Details of the ABE9 base editor are described in International PCT Application No. PCT / 2020 / 049975, which is incorporated herein by reference in its entirety.

[0075] "Adenosine deaminase base editor 9 (ABE9) polypeptide" means a polynucleotide encoding ABE9.

[0076] As used herein, "administering" refers to providing a patient or subject with one or more of the compositions described herein. By way of example and not limitation, administration of a composition (e.g., injection) 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, depot injection or by progressive infusion over time. In some embodiments, parenteral administration includes intravascular, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transorgan, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, and intrasternal infusion or injection. Alternatively or concurrently, administration can be by the oral route.

[0077] "Agent" means any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0078] "Alteration" means a change (e.g., increase or decrease) in the structure, expression level, or activity of a gene or polypeptide detected by standard methods known in the art such as those described herein. As used herein, an alteration (e.g., increase or decrease) includes a change in a polynucleotide or polypeptide or a change in the expression level, such as a 10% change, 25% change, 40% change, or 50% change or higher.

[0079] As used herein, "allogeneic" refers to cells of the same species that are genetically different from the cells being compared.

[0080] "Alleviate" means to reduce, suppress, attenuate, eliminate, prevent the development or progression of a disease or to stabilize the development or progression of a disease.

[0081] "Analogue" means a molecule that is not identical but has similar functional or structural characteristics. For example, a polynucleotide or polypeptide analogue retains the biological activity of the corresponding naturally occurring polynucleotide or polypeptide while having certain modifications that enhance the function of the analogue relative to the naturally occurring polynucleotide or polypeptide. Such modifications can increase the affinity of the analogue for DNA, efficiency, specificity, protease or protease resistance, membrane permeability, and / or half-life without altering, for example, ligand binding. Analogues can include non-natural nucleotides or amino acids.

[0082] "Antitumorigenic activity" means preventing or inhibiting the mutation and / or proliferation of tumors.

[0083] As used herein, "autologous" refers to cells from the same subject.

[0084] 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, tetra-specific antibodies, diabodies, triabodies, and tetra-bodies), and antigen-binding fragments of antibodies, including, for example, Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments. Unless otherwise indicated, the term "monoclonal antibody" (mAb) is meant to include both the intact molecule and antibody fragments capable of specifically binding to the target protein (including, for example, Fab and F(ab')2 fragments). As used herein, Fab and F(ab')2 fragments refer to antibody fragments that lack the Fc fragment of the intact antibody. These antibody fragments are exemplified herein.

[0085] "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 with NCBI accession number NP_001183 or a fragment thereof. Exemplary BCMA polypeptide sequences are provided below.

[0086] >NP_001183.2 Tumor necrosis factor receptor superfamily member 17 [Homo sapiens]

[0087] MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR

[0088] This antigen can be targeted in the treatment of relapsed or refractory multiple myeloma and other hematological malignancies.

[0089] "B-cell maturation antigen, or tumor necrosis factor receptor superfamily member 17, (BCMA) polynucleotide" means a nucleic acid molecule encoding a BCMA polypeptide. The BCMA gene encodes a cell surface antigen that recognizes B-cell activating factor. Exemplary B2M polynucleotide sequences are provided below.

[0090] >NM_001192.2 Homo sapiens TNF receptor superfamily member 17 (TNFRSF17), mRNA

[0091] AAGACTCAAACTTAGAAACTTGAATTAGATGTGGTATTCAAATCCTTAGCTGCCGCGAAGACACAGACAGCCCCCGTAAGAACCCACGAAGCAGGCGAAGTTCATTGTTCTCAACATTCTAGCTGCTCTTGCTGCATTTGCTCTGGAATTCTTGTAGAGATATTACTTGTCCTTCCAGGCTGTTCTTTCTGTAGCTCCCTTGTTTTCTTTTTGTGATCATGTTGCAGATGGCTGGGCAGTGCTCCCAAAATGAATATTTTGACAGTTTGTTGCATGCTTGCATACCTTGTCAACTTCGATGTTCTTCTAATACTCCTCCTCTAACATGTCAGCGTTATTGTAATGCAAGTGTGACCAATTCAGTGAAAGGAACGAATGCGATTCTCTGGACCTGTTTGGGACTGAGCTTAATAATTTCTTTGGCAGTTTTCGTGCTAATGTTTTTGCTAAGGAAGATAAACTCTGAACCATTAAAGGACGAGTTTAAAAACACAGGATCAGGTCTCCTGGGCATGGCTAACATTGACCTGGAAAAGAGCAGGACTGGTGATGAAATTATTCTTCCGAGAGGCCTCGAGTACACGGTGGAAGAATGCACCTGTGAAGACTGCATCAAGAGCAAACCGAAGGTCGACTCTGACCATTGCTTTCCACTCCCAGCTATGGAGGAAGGCGCAACCATTCTTGTCACCACGAAAACGAATGACTATTGCAAGAGCCTGCCAGCTGCTTTGAGTGCTACGGAGATAGAGAAATCAATTTCTGCTAGGTAATTAACCATTTCGACTCGAGCAGTGCCACTTTAAAAATCTTTTGTCAGAATAGATGATGTGTCAGATCTCTTTAGGATGACTGTATTTTTCAGTTGCCGATACAGCTTTTTGTCCTCTAACTGTGGAAACTCTTTATGTTAGATATATTTCTCTAGGTTACTGTTGGGAGCTTAATGGTAGAAACTTCCTTGGTTTCATGATTAAACTCTTTTTTTTCCTGA

[0092] "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 cytidine 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 cytidine within DNA. In some embodiments, the base editor is a fusion protein comprising a cytidine deaminase or an adenosine deaminase. In some embodiments, the base editor is a Cas9 protein fused to a cytidine deaminase or an adenosine deaminase. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a cytidine deaminase or an adenosine deaminase. In some embodiments, the base editor is fused to an inhibitor of base excision repair, e.g., the 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 the UG domain.

[0093] In some embodiments, a cytidine deaminase or adenosine deaminase nucleobase editor is a polypeptide comprising the following domains A-B: NH2-[A-B]-COOH, wherein A comprises a cytidine 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 cytidine or adenosine deaminase nucleobase editor polypeptide of the previous aspect contains: NH2-[A n -B o -COOH, wherein A comprises a cytidine deaminase domain, an adenosine deaminase domain or an active fragment thereof, where 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.

[0094] In some embodiments, the base editor is a cytidine 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 cytidine base editor (CBE). In some embodiments, the base editor is 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 Cas9 is known in the art and described, for example, in 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., the UGI domain or the dISN domain. In some embodiments, the fusion protein comprises a Cas9 nickase that is fused to a deaminase and an inhibitor of base excision repair, such as the UGI or dISN domain. In other embodiments, the base editor is a base - free base editor.

[0095] 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 catalytically - dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is 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.

[0096] In some embodiments, the base editor is generated by cloning an adenosine deaminase variant (e.g., TadA*7.10) into a scaffold that comprises 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 that comprises circular permutant Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear localization sequence. Circular permutant Cas9 is known in the art and described, for example, in Oakes et al., Cell 176, 254–267, 2019.

[0097] 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 catalytically dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is 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 uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of base excision repair is inosine base excision repair inhibitor.

[0098] 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 herein by reference 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: eaao4774 (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 herein by reference.

[0099] For example, an adenosine base editor (ABE) used in the base editing compositions, systems, and methods described herein has the nucleic acid sequence (8877 base pairs) 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.). Polynucleotide sequences having at least 95% or higher identity to the ABE nucleic acid sequence are also encompassed.

[0100]

[0101] In some embodiments, the base editor is adenosine deaminase base editor 8 (ABE8). In some embodiments, ABE8 is selected from the base editors in Tables 13, 14, or 16 below. In some embodiments, ABE8 contains an adenosine deaminase evolved from TadA. In some embodiments, the adenosine deaminase variant of ABE8 is the TadA*8 variant as described in Tables 11, 13, or 14 below. In some embodiments, the adenosine deaminase is the TadA*7.10 variant (e.g., TadA*8), which comprises one or more of the following selected changes: Y147T, Q154S, Y123H, V82S, T166R, and / or Q154R. In various embodiments, ABE8 comprises the TadA*7.10 variant (e.g., TadA*8), which has a combination of changes selected from the following groups: 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, ABE8 is a monomeric construct. In some embodiments, ABE8 is a heterodimeric construct. In some embodiments, the ABE8 base editor comprises the sequence:

[0102] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0103] 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 (8,877 base pairs) provided below (Addgene, Watertown, MA.; Komor AC, et al., 2017, Sci Adv., 30; 3(8): eaao4774. doi: 10.1126 / sciadv.aao4774). Polynucleotide sequences having at least 95% or higher identity to the BE4 nucleic acid sequence are also encompassed.

[0104]

[0105]

[0106]

[0107] In some embodiments, the cytidine base editor is BE4 having a nucleic acid sequence selected from one of the following:

[0108] Original BE4 nucleic acid sequence:

[0109]

[0110] BE4 codon-optimized 1 nucleic acid sequence:

[0111]

[0112] BE4 codon-optimized 2 nucleic acid sequence:

[0113]

[0114] "Base editing activity" means acting to chemically modify a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, e.g., converting a target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g., converting a target A·T to G·C. In another embodiment, the base editing activity is cytidine deaminase activity, e.g., converting a target C·G to T·A, and adenosine or adenine deaminase activity, e.g., converting a target A·T to G·C.

[0115] In some embodiments, base editing activity is evaluated 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 a 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 a base editor when base editing is performed in a cell population.

[0116] The term "base editor system" refers to a system for editing the nucleobases 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 for deaminating the nucleobase (e.g., adenosine deaminase or cytidine deaminase); and (3) one or more guide polynucleotides (e.g., guide RNA). 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 adenosine deaminase base editor 8 (ABE8).

[0117] 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 heterologous 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.

[0118] In some embodiments, a base editor system can include more than one base editing component. For example, a base editor system can include more than one deaminase. In some embodiments, a base editor can include one or more cytidine deaminases. In some embodiments, a base editor 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.

[0119] The nucleobase component of the base editor system and the polynucleotide programmable nucleotide binding component can be associated with each other covalently or non-covalently, or any combination of their association and interaction. 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 may be capable of binding, interacting, associating, or forming a complex with a polypeptide. In some embodiments, the additional heterologous moiety may be capable of binding, interacting, associating, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous moiety may 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, an MS2 coat protein domain, a PP7 coat protein domain, an SfMuCom coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0120] The base editor system can further comprise a guide polynucleotide component. It should be understood that the components of the base editor system can associate with each other via covalent bonds, non-covalent interactions, or any combination of their associations and interactions. In some embodiments, the deaminase 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, such as a 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 may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous moiety may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to the guide polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous moiety may 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, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0121] In some embodiments, the base editor system may further comprise an inhibitor component of base excision repair (BER). It should be understood that the components of the base editor system may associate with each other via covalent bonds, non-covalent interactions, or any combination of their associations and interactions. The inhibitor component of BER may comprise a BER inhibitor. In some embodiments, the inhibitor of BER may be a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of BER may be an inosine BER inhibitor. In some embodiments, the inhibitor of BER may be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain may be fused or linked to the inhibitor of BER. In some embodiments, the polynucleotide programmable nucleotide binding domain may be fused or linked to a deaminase domain and an inhibitor of BER. In some embodiments, the polynucleotide programmable nucleotide binding domain may 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 component of BER may 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.

[0122] In some embodiments, the inhibitor of BER may be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the inhibitor component of BER of the base editor system may 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 of the guide polynucleotide (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) may be fused or linked to the inhibitor of BER. In some embodiments, the additional heterologous moiety may be capable of binding, interacting, associating, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to the guide polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous moiety may be capable of binding to a polynucleotide linker. The additional heterologous moiety may be a protein domain. In some embodiments, the additional heterologous moiety may be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0123] "β-2 microglobulin (B2M) polypeptide" means a protein that has at least about 85% amino acid sequence identity with UniProt accession number P61769 or a fragment thereof and has immunomodulatory activity. Exemplary B2M polypeptide sequences are provided below.

[0124] >sp|P61769|B2MG_HUMAN β-2-microglobulin OS=Homo sapiens OX=9606 GN=B2M PE=1 SV=1

[0125] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

[0126] "β-2 microglobulin (B2M) polynucleotide" means a nucleic acid molecule that encodes a B2M polypeptide. The β-2 microglobulin gene encodes a serum protein associated with the major histocompatibility complex. B2M is involved in non-self recognition by host CD8+ T cells. Exemplary B2M polynucleotide sequences are provided below.

[0127] >DQ217933.1 Homo sapiens β-2 microglobulin (B2M) gene, complete coding sequence

[0128]

[0129] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease or a fragment thereof that comprises a Cas9 protein (e.g., a protein comprising the DNA cleavage domain of Cas9 that is active, inactive, or partially active and / or the gRNA-binding domain of Cas9). Cas9 nuclease sometimes refers to Casn1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). A CRISPR cluster contains spacer sequences, sequences complementary to precursor mobile elements, and target invading nucleic acids. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, proper processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), the endogenous ribonuclease 3 (rnc), and the Cas9 protein. TracrRNA serves as a guide for ribonuclease 3-assisted pre-crRNA processing. Subsequently, the Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer sequence. The target strand that is not complementary to the crRNA is first endonucleolytically cleaved and then exonucleolytically trimmed 3'-5'. In nature, DNA binding and cleavage generally require a protein and two RNAs. However, a single guide RNA ("sgRNA" or simply "gRNA") can be engineered to incorporate multiple crRNAs and tracrRNAs into a single RNA species. See, e.g., Jinek M. et al., Charpentier E. Science 337:816-821 (2012), the entire content of which is incorporated herein by reference. Cas9 recognizes a short motif (PAM or protospacer adjacent motif) in the CRISPR repeat to help distinguish self from non-self.The Cas9 nuclease sequence and structure are well-known to those skilled in the art (see, e.g., “Complete genome sequence of an M1 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 (2011); 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 various species, including Streptococcus pyogenes and Streptococcus thermophilus. Based on the present disclosure, other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art, 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).

[0130] A nuclease - inactive Cas9 protein can be interchangeably referred to as a “dCas9” protein (for “dead” Cas9 with respect to nuclease) or catalytically inactive Cas9. Cas9 proteins (or fragments thereof) for generating an inactive DNA - cleavage domain 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 respective entire contents of which are incorporated herein by reference). For example, the DNA - cleavage domain of Cas9 is known to include two sub - domains: the HNH nuclease sub - domain and the RuvC1 sub - domain. The HNH sub - domain cleaves the strand complementary to the gRNA, while the RuvC1 sub - domain cleaves the non - complementary strand. Mutations in these sub - domains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337:816 - 821 (2012); Qi et al., Cell. 28; 152(5):1173 - 83 (2013)). In some embodiments, dCas9 corresponds to or partially or wholly comprises a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. In some embodiments, the dCas9 domain contains the D10A and H840A mutations or corresponding mutations in another Cas9. In some embodiments, the Cas9 nuclease has an inactivated (e.g., inactivated) DNA - cleavage domain, in other words, Cas9 is a nickase, referred to as an “nCas9” protein (for “nickase” Cas9). It should be understood that other Cas9 proteins (e.g., nuclease - dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease - active Cas9), including their variants and homologs, are within the scope of the present disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided herein. In some embodiments, the Cas9 protein is nuclease - dead Cas9 (dCas9). In some embodiments, the Cas9 protein is Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is nuclease - active Cas9.

[0131] 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) the gRNA-binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, a 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%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identical to wild-type Cas9. In some embodiments, compared to wild-type Cas9, a Cas9 variant may 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. In some embodiments, a Cas9 variant comprises a fragment of Cas9 (e.g., the gRNA-binding domain or the DNA cleavage domain) such that the fragment is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, 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% the length of the amino acids of the corresponding wild-type Cas9.

[0132] 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.

[0133] In some embodiments, Cas9 refers to 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 refers to Cas9 from any other organism.

[0134] In some embodiments, Cas9 is from Neisseria meningitidis (Nme). In some embodiments, 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)).

[0135] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas9 protein (e.g., one of the Cas9 sequences provided herein). However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but only one or more fragments thereof. For example, in some embodiments, the Cas9 fusion proteins provided herein comprise a Cas9 fragment that binds crRNA and tracrRNA or sgRNA, but does not comprise a functional nuclease domain, e.g., in cases where it only comprises a truncated version of the nuclease domain or no nuclease domain at all.

[0136] 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.

[0137] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., Nanoarchaeum), which constitutes the domain and kingdom of single-celled prokaryotic microorganisms. In some embodiments, Cas9 refers to CasX or CasY, which have been described, for example, in Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb 21. doi:10.1038 / cr.2017.21, the entire content of which is incorporated herein by reference. Using genome-resolved metagenomics, a large number of CRISPR-Cas systems have been identified, including Cas9 reported for the first time in the archaeal domain of life. Such diverse Cas9 proteins were found as active CRISPR-Cas systems in the little-studied Nanoarchaeum. Two previously unknown systems, CRISPR-CasX and CRISPR-CasY, were found in bacteria and are among the most compact systems discovered to date. 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 should be understood that other RNA-guided DNA-binding proteins can be used as nucleic acid programmable DNA-binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0138] In certain embodiments, the napDNAbps useful in the methods of the invention include circular permutants, which are known in the art and are described, for example, in Oakes et al., Cell 176, 254–267, 2019.

[0139] Non-limiting examples of polynucleotide programmable nucleotide-binding domains that can be incorporated into base editors include domains derived from CRISPR proteins, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0140] 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 should be understood that CasX and CasY from other bacterial species can also be used according to the present disclosure.

[0141] The term "Cas12b" or "Cas12b domain" refers to an RNA-guided nuclease or a fragment thereof that comprises Cas12b, a C2c1 protein (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas12b and / or a gRNA-binding domain of Cas12b). Their respective contents 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. Based on the present invention, other suitable Cas12b nuclease sequences will be apparent to those skilled in the art.

[0142] 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, compared to wild-type Cas12b, a Cas12b variant may 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. 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 listed herein.

[0143] The "Cbl proto-oncogene B (CBLB) polypeptide" means a protein that has at least about 85% amino acid sequence identity with GenBank accession number ABC86700.1 or a fragment thereof and is involved in the regulation of the immune response. Exemplary CBLB polypeptide sequences are provided below.

[0144] >ABC86700.1 CBL-B[Homo sapiens]

[0145] MANSMNGRNPGGRGGNPRKGRILGIIDAIQDAVGPPKQAAADRRTVEKTWKLMDKVVRLCQNPKLQLKNSPPYILDILPDTYQHLRLILSKYDDNQKLAQLSENEYFKIYIDSLMKKSKRAIRLFKEGKERMYEEQSQDRRNLTKLSLIFSHMLAEIKAIFPNGQFQGDNFRITKADAAEFWRKFFGDKTIVPWKVFRQCLHEVHQISSGLEAMALKSTIDLTCNDYISVFEFDIFTRLFQPWGSILRNWNFLAVTHPGYMAFLTYDEVKARLQKYSTKPGSYIFRLSCTRLGQWAIGYVTGDGNILQTIPHNKPLFQALIDGSREGFYLYPDGRSYNPDLTGLCEPTPHDHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTAWQESDGQGCPFCRCEIKGTEPIIVDPFDPRDEGSRCCSIIDPFGMPMLDLDDDDDREESLMMNRLANVRKCTDRQNSPVTSPGSSPLAQRRKPQPDPLQIPHLSLPPVPPRLDLIQKGIVRSPCGSPTGSPKSSPCMVRKQDKPLPAPPPPLRDPPPPPPERPPPIPPDNRLSRHIHHVESVPSRDPPMPLEAWCPRDVFGTNQLVGCRLLGEGSPKPGITASSNVNGRHSRVGSDPVLMRKHRRHDLPLEGAKVFSNGHLGSEEYDVPPRLSPPPPVTTLLPSIKCTGPLANSLSEKTRDPVEEDDDEYKIPSSHPVSLNSQPSHCHNVKPPVRSCDNGHCMLNGTHGPSSEKKSNIPDLSIYLKGDVFDSASDPVPLPPARPPTRDNPKHGSSLNRTPSDYDLLIPPLGEDAFDALPPSLPPPPPPARHSLIEHSKPPGSSSRPSSGQDLFLLPSDPFVDLASGQVPLPPARRLPGENVKTNRTSQDYDQLPSCSDGSQAPARPPKPRPRRTAPEIHHRKPHGPEAALENVDAKIAKLMGEGYAFEEVKRALEIAQNNVEVARSILREFAFPPPVSPRLNL

[0146] "Cbl proto-oncogene B (CBLB) polynucleotide" means a nucleic acid molecule encoding a CBLB polypeptide. The CBLB gene encodes an E3 ubiquitin ligase. Exemplary CBLB nucleic acid sequences are provided below.

[0147]

[0148] "Chimeric antigen receptor" or "CAR" means a synthetic or engineered receptor that comprises an extracellular antigen-binding domain that confers antigen specificity on an immune effector cell and that is joined to one or more intracellular signaling domains (e.g., a T cell signaling domain). In some embodiments, the CAR includes a transmembrane domain.

[0149] "Chimeric antigen receptor T cell" or "CAR-T cell" means a T cell that expresses a CAR and that has antigen specificity determined by the domain of the CAR that is derived from an antibody. As used herein, "CAR-T cell" includes a T cell or an NK cell. As used herein, "CAR-T cell" includes a cell that has been engineered to express a CAR or a T cell receptor (TCR). In some embodiments, the CAR-T cell can be a T helper CD4+ and / or a T effector CD8+ cell, optionally present in the defined ratios. Methods for preparing 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 Publication WO2012 / 079000, WO2013 / 059593, and U.S. Patent Publication 2012 / 0213783, each of which is incorporated herein by reference in its entirety).

[0150] "Class II major histocompatibility complex transactivator (CIITA)" means a protein that has at least about 85% amino acid sequence identity with NCBI accession number NP_001273331.1 or a fragment thereof and that has immunomodulatory activity. Exemplary amino acid sequences are provided below.

[0151] >NP_001273331.1 Class II MHC transactivator isoform 1 [Homo sapiens]

[0152]

[0153] "Class II major histocompatibility complex transactivator (CIITA)" means a nucleic acid encoding a CIITA polypeptide. Exemplary CIITA nucleic acid sequences are provided below.

[0154] >NM_001286402.1 Homo sapiens Class II major histocompatibility complex transactivator (CIITA), transcript variant 1, mRNA

[0155]

[0156] In the present disclosure, terms such as "comprising", "comprises", "containing", and "having" may have the meanings as defined by the United States Patent Law and may mean "including", "including within", etc.; terms such as "consisting essentially of" or "consisting mainly of" have the meanings as defined by the United States Patent Law, and such terms are open-ended, allowing the presence of elements in excess of those recited, provided that the presence of elements in excess of those recited does not change the basic or novel characteristics of those recited, but excluding prior art embodiments.

[0157] "Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) polypeptide" means a protein that has at least about 85% sequence identity with NCBI accession number EAW70354.1 or a fragment thereof. Exemplary amino acid sequences are provided below:

[0158] >EAW70354.1 Cytotoxic T-lymphocyte-associated protein 4 [Homo sapiens]

[0159] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN

[0160] "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 inhibitory signals to T cells. Exemplary CTLA-4 nucleic acid sequences are provided below.

[0161] >BC074842.2 Homo sapiens cytotoxic T-lymphocyte-associated protein 4, mRNA (cDNA clone MGC:104099 IMAGE:30915552), complete coding sequence

[0162] GACCTGAACACCGCTCCCATAAAGCCATGGCTTGCCTTGGATTTCAGCGGCACAAGGCTCAGCTGAACCTGGCTACCAGGACCTGGCCCTGCACTCTCCTGTTTTTTCTTCTCTTCATCCCTGTCTTCTGCAAAGCAATGCACGTGGCCCAGCCTGCTGTGGTACTGGCCAGCAGCCGAGGCATCGCCAGCTTTGTGTGTGAGTATGCATCTCCAGGCAAAGCCACTGAGGTCCGGGTGACAGTGCTTCGGCAGGCTGACAGCCAGGTGACTGAAGTCTGTGCGGCAACCTACATGATGGGGAATGAGTTGACCTTCCTAGATGATTCCATCTGCACGGGCACCTCCAGTGGAAATCAAGTGAACCTCACTATCCAAGGACTGAGGGCCATGGACACGGGACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATACTACCTGGGCATAGGCAACGGAACCCAGATTTATGTAATTGATCCAGAACCGTGCCCAGATTCTGACTTCCTCCTCTGGATCCTTGCAGCAGTTAGTTCGGGGTTGTTTTTTTATAGCTTTCTCCTCACAGCTGTTTCTTTGAGCAAAATGCTAAAGAAAAGAAGCCCTCTTACAACAGGGGTCTATGTGAAAATGCCCCCAACAGAGCCAGAATGTGAAAAGCAATTTCAGCCTTATTTTATTCCCATCAATTGAGAAACCATTATGAAGAAGAGAGTCCATATTTCAATTTCCAAGAGCTGAGG

[0163] "Cluster of Differentiation 2 (CD2) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI accession number NP_001758.2 or a fragment thereof and has immunomodulatory activity. Exemplary amino acid sequences are provided below.

[0164] >NP_001758.2 T-cell surface antigen CD2 isoform 2 precursor [Homo sapiens]

[0165]

[0166] 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 in.

[0167] "Cluster of Differentiation 2 (CD2) polynucleotide" means a nucleic acid encoding a CD2 polypeptide. Exemplary CD2 nucleic acid sequences are provided below. >NM_001767.5 Homo sapiens CD2 molecule (CD2), transcript variant 2, RNA

[0168]

[0169] "Cluster of Differentiation 5 (CD5) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI accession number NP_001333385.1 or a fragment thereof and has immunomodulatory activity. Exemplary amino acid sequences are provided below.

[0170] >NP_001333385.1 T-cell surface glycoprotein CD5 isoform 2 [Homo sapiens]

[0171] MVCSQSWGRSSKQWEDPSQASKVCQRLNCGVPLSLGPFLVTYTPQSSIICYGQLGSFSNCSHSRNDMCHSLGLTCLEPQKTTPPTTRPPPTTTPEPTAPPRLQLVAQSGGQHCAGVVEFYSGSLGGTISYEAQDKTQDLENFLCNNLQCGSFLKHLPETEAGRAQDPGEPREHQPLPIQWKIQNSSCTSLEHCFRKIKPQKSGRVLALLCSGFQPKVQSRLVGGSSICEGTVEVRQGAQWAALCDSSSARSSLRWEEVCREQQCGSVNSYRVLDAGDPTSRGLFCPHQKLSQCHELWERNSYCKKVFVTCQDPNPAGLAAGTVASIILALVLLVVLLVVCGPLAYKKLVKKFRQKKQRQWIGPTGMNQNMSFHRNHTATVRSHAENPTASHVDNEYSQPPRNSHLSAYPALEGALHRSSMQPDNSSDSDYDLHGAQRL

[0172] "Cluster of Differentiation 5 (CD5) polynucleotide" means a nucleic acid encoding a CD5 polypeptide. Exemplary CD5 nucleic acid sequences are provided below.

[0173] >NM_001346456.1 Homo sapiens CD5 molecule (CD5), transcript variant 2, mRNA

[0174]

[0175]

[0176] "Cluster of Differentiation 7 (CD7) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI Reference Sequence NP_006128.1 or a fragment thereof and has immunomodulatory activity. Exemplary amino acid sequences are provided below.

[0177] >NP_006128.1 T cell antigen precursor [Homo sapiens]

[0178]

[0179] "Cluster of Differentiation 7 (CD7) polynucleotide" means a nucleic acid molecule that encodes a CD7 polypeptide. Exemplary CD7 nucleic acid sequences are provided below.

[0180] >NM_006137.7 Homo sapiens CD7 molecule (CD7), mRNA

[0181]

[0182] "Cluster of Differentiation 33 (CD33) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI Reference Sequence NP_001763.3 or a fragment thereof. CD33 is also known as Siglec-3. Exemplary amino acid sequences are provided below.

[0183] >NP_001763.3 Myeloid cell surface antigen CD33 isoform 1 precursor [Homo sapiens]

[0184]

[0185] "Cluster of Differentiation 33 (CD33) polynucleotide" means a nucleic acid molecule that encodes a CD33 polypeptide. Exemplary CD33 nucleic acid sequences are provided below.

[0186] >NM_001772.4 Homo sapiens CD33 molecule (CD33), transcript variant 1, mRNA

[0187]

[0188] "Cluster of Differentiation 52 (CD52) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI Reference Sequence NP_001794.2 or a fragment thereof. CD52 is also known as CAMPATH-1. Exemplary amino acid sequences are provided below.

[0189] >NP_001794.2 CAMPATH-1 antigen precursor [Homo sapiens]

[0190] 1 MKRFLFLLLTISLLVMVQIQTGLSGQNDTSQTSSPSASSNISGGIFLFFVANAIIHLFCF

[0191] 61 S

[0192] "Cluster of Differentiation 52 (CD52) polynucleotide" means a nucleic acid molecule that encodes a CD52 polypeptide. Exemplary CD52 nucleic acid sequences are provided below.

[0193] >NM_001803.3 Homo sapiens CD52 molecule (CD52), mRNA

[0194]

[0195] "Cluster of Differentiation 123 (CD123) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI Reference Sequence NP_002174.1 or a fragment thereof. CD123 is also known as interleukin-3 receptor. Exemplary amino acid sequences are provided below.

[0196] >NP_002174.1 Interleukin-3 receptor subunit alpha isoform 1 precursor [Homo sapiens]

[0197]

[0198] "Cluster of Differentiation 123 (CD123) polynucleotide" means a nucleic acid molecule that encodes a CD123 polypeptide. Exemplary CD123 nucleic acid sequences are provided below.

[0199] >NM_002183.4 Homo sapiens interleukin 3 receptor subunit alpha (IL3RA), transcript variant 1, mRNA

[0200]

[0201] "Cluster of Differentiation 137 (CD137) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI reference sequence NP_001552.2 or a fragment thereof. Cd137 is also known as 4-1BB. Exemplary amino acid sequences are provided below.

[0202] >NP_001552.2 Tumor necrosis factor receptor superfamily member 9 precursor [Homo sapiens]

[0203]

[0204] "Cluster of Differentiation 137 (CD137) polynucleotide" means a nucleic acid molecule that encodes a CD137 polypeptide. Exemplary CD137 nucleic acid sequences are provided below.

[0205] >NM_001561.6 Homo sapiens TNF receptor superfamily member 9 (TNFRSF9), mRNA

[0206]

[0207]

[0208] "Cluster of Differentiation 247 (CD247) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI reference sequence NP_932170.1 or a fragment thereof. CD137 is also known as CD3ζ. Exemplary amino acid sequences are provided below.

[0209] >NP_932170.1 T-cell surface glycoprotein CD3ζ chain isoform 1 precursor [Homo sapiens]

[0210]

[0211] "Cluster of Differentiation 247 (CD247) polynucleotide" means a nucleic acid molecule that encodes a CD247 polypeptide. Exemplary CD247 nucleic acid sequences are provided below.

[0212] >NM_NM_198053.3 Homo sapiens CD247 molecule (CD247), transcript variant 1, mRNA

[0213]

[0214] "Co-administration" or "co-administering" means administering two or more therapeutic agents or pharmaceutical compositions during a course of treatment. The co-administration can be simultaneous or sequential. Sequential administration of a later-administered therapeutic agent or pharmaceutical composition can occur at any time during the course of treatment after administration of the first pharmaceutical composition or therapeutic agent.

[0215] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid having common properties. A functional way to define the common properties between 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)). Based on such analysis, multiple groups of amino acids can be defined, where amino acids within a group preferentially exchange with each other and thus are most similar to each other in terms of their effect on the overall protein structure (Schulz, G.E. and Schirmer, R.H., ibid.). Non-limiting examples of conservative mutations include amino acid substitutions of amino acids such as lysine for arginine and vice versa so that the positive charge can be maintained; glutamic acid for aspartic acid and vice versa so that the negative charge can be maintained; serine for threonine so that the free –OH can be maintained; and glutamine for asparagine so that the free –NH2 can be maintained.

[0216] The terms "coding sequence" or "protein-coding sequence", which may be used interchangeably herein, refer to a polynucleotide segment that encodes a protein. This region or sequence is bounded by a start codon near the 5'-end and a stop codon near the 3'-end. The coding sequence can also be referred to as an open reading frame.

[0217] "Codon optimization" means the process of modifying a nucleic acid sequence by replacing at least one codon of a native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with a codon that is more frequently or most frequently used in the genes of the host cell of interest while maintaining the native amino acid sequence. For certain codons of a particular amino acid, various species exhibit a particular bias. Codon bias (differences in codon usage between organisms) is often related to the translational efficiency of messenger RNA (mRNA), which is believed to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons most frequently 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 various 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 for codon optimizing a particular sequence for expression in a particular host cell are also available, 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 the sequence encoding an engineered nuclease correspond to the codons most frequently used for a particular amino acid.

[0218] "Cytidine deaminase" means a polypeptide or a fragment thereof that is capable of catalyzing a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, the cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. PmCDA1 (Petromyzon marinus cytidine deaminase 1, ("PmCDA1")) from the sea lamprey (Petromyzon marinus), or AID (activation-induced cytidine deaminase; ("AICDA")) and APOBEC from mammals (e.g., human, pig, cow, horse, monkey, etc.) are exemplary cytidine deaminases.

[0219] The nucleotide sequences and amino acid sequences of PmCDA1 and the nucleotide sequences and amino acid sequences of human AID are shown below.

[0220] >tr|A5H718|A5H718_PETMA Cytidine deaminase OS=Sea lamprey (Petromyzon marinus) OX=7757 PE=2 SV=1

[0221] MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV

[0222] >EF094822.1 Sea lamprey isolated PmCDA.21 Cytidine deaminase mRNA, complete cds

[0223] TGACACGACACAGCCGTGTATATGAGGAAGGGTAGCTGGATGGGGGGGGGGGGAATACGTTCAGAGAGGACATTAGCGAGCGTCTTGTTGGTGGCCTTGAGTCTAGACACCTGCAGACATGACCGACGCTGAGTACGTGAGAATCCATGAGAAGTTGGACATCTACACGTTTAAGAAACAGTTTTTCAACAACAAAAAATCCGTGTCGCATAGATGCTACGTTCTCTTTGAATTAAAACGACGGGGTGAACGTAGAGCGTGTTTTTGGGGCTATGCTGTGAATAAACCACAGAGCGGGACAGAACGTGGAATTCACGCCGAAATCTTTAGCATTAGAAAAGTCGAAGAATACCTGCGCGACAACCCCGGACAATTCACGATAAATTGGTACTCATCCTGGAGTCCTTGTGCAGATTGCGCTGAAAAGATCTTAGAATGGTATAACCAGGAGCTGCGGGGGAACGGCCACACTTTGAAAATCTGGGCTTGCAAACTCTATTACGAGAAAAATGCGAGGAATCAAATTGGGCTGTGGAACCTCAGAGATAACGGGGTTGGGTTGAATGTAATGGTAAGTGAACACTACCAATGTTGCAGGAAAATATTCATCCAATCGTCGCACAATCAATTGAATGAGAATAGATGGCTTGAGAAGACTTTGAAGCGAGCTGAAAAACGACGGAGCGAGTTGTCCATTATGATTCAGGTAAAAATACTCCACACCACTAAGAGTCCTGCTGTTTAAGAGGCTATGCGGATGGTTTTC

[0224] >tr|Q6QJ80|Q6QJ80_Human activation-induced cytidine deaminase OS=Homo sapiens OX=9606 GN=AICDA PE=2 SV=1

[0225] MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKAPV

[0226]

[0227] 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 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).

[0228] Other exemplary deaminases that can be fused to Cas9 according to aspects of the present disclosure are provided herein. It should be understood that in some embodiments, the active domain of the corresponding sequence can be used, such as a domain without a localization signal (nuclear localization sequence, without a nuclear export signal, cytoplasmic localization signal).

[0229] 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, 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 bacteria. In some embodiments, the adenosine deaminase is from bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Caulobacter crescentus.

[0230] 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.

[0231] For example, base editors are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), which are hereby incorporated by reference in their 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:eaao4774 (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.

[0232] "Detecting" means identifying the presence, absence, or amount of an analyte to be detected.

[0233] "Detectable label" means a composition that is linked to a molecule of interest such that the latter can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, available labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in enzyme-linked immunosorbent assay (ELISA)), biotin, digoxin, or haptens.

[0234] "Disease" means any disorder or lesion that impairs or interferes with the normal function of cells, tissues, or organs. In one embodiment, the disease is neoplasia or cancer. In some embodiments, the disease is a hematological cancer. "Hematological cancer" means a malignant disorder of the cells of the immune system. In some embodiments, the hematological cancer is leukemia, myeloma, and / or lymphoma. Lymphoma and leukemia are examples of "liquid cancers" or cancers that exist in the blood and result from the transformation of hematopoietic precursors in the bone marrow or mature hematopoietic cells in the blood. Leukemia can be lymphocytic or myelogenous and acute or chronic. In the case of myeloma, the transformed cells are fully differentiated plasma cells, which may exist as a dispersed collection of malignant cells or as a solid mass in the bone marrow. In the case of lymphoma, transformed lymphocytes in secondary lymphoid tissue give rise to solid masses. Lymphomas are classified as Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL).

[0235] In some embodiments, the hematological cancer is a B-cell cancer. In some embodiments, the B-cell cancer is lymphoma or leukemia. In some cases, leukemia includes preleukemia. In some cases, leukemia is acute leukemia. Acute leukemia includes, for example, acute myeloid leukemia (AML). Acute leukemia also includes, for example, acute lymphoblastic leukemia or acute lymphocytic leukemia (ALL); ALL includes B-lineage ALL, T-lineage ALL, and T-cell acute lymphoblastic leukemia (T-ALL).

[0236] Non-limiting examples of the disease include 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 disease, extranodal NK / T-cell lymphoma, adult T-cell leukemia / lymphoma, T-cell prolymphocytic leukemia, subcutaneous panniculitis-like T-cell lymphoma, primary cutaneous gamma / delta T-cell lymphoma, aggressive NK-cell leukemia, 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).

[0237] As used herein, the term "effective amount" refers to an amount of a bioactive agent sufficient to elicit a desired biological response. In some embodiments, the effective amount is an amount relative to that required to alleviate the symptoms of a disease in an untreated patient. The effective amount of an active agent used to therapeutically treat a disease in practicing the present invention varies depending on the mode of administration and the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. This amount is referred to as an "effective" amount. In one embodiment, the effective amount is an amount of a base editor of the present invention (e.g., a fusion protein comprising a programmable DNA-binding protein, a nucleobase editor, and a gRNA) sufficient to introduce a change into a gene of interest in a cell (e.g., an in vitro cell or an in vivo cell). In one embodiment, the effective amount is the amount of base editor required to achieve a therapeutic effect (e.g., alleviating or controlling a disease or its symptoms or conditions). Such a therapeutic effect need not be sufficient to alter the gene of interest in all cells of a subject, tissue, or organ, but may only alter the gene of interest in about 1%, 5%, 10%, 25%, 50%, 75%, or more of the cells present in a subject, tissue, or organ.

[0238] In some embodiments, the effective amount of a fusion protein provided herein, e.g., the effective amount of a nucleobase editor comprising an 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, nuclease, hybrid protein, protein dimer, complex of a protein (or protein dimer) and a polynucleotide, or 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.

[0239] As used herein, the term "epitope" means an antigenic determinant. An epitope is a part of an antigen molecule that determines the specific antibody that will recognize and bind to it by its structure.

[0240] "Fas cell surface death receptor (FAS) polypeptide" means a protein having at least about 85% amino acid sequence identity with NCBI accession number NP_000034.1 or a fragment thereof. Exemplary amino acid sequences are provided below.

[0241] >NP_000034.1 Tumor necrosis factor receptor superfamily member 6 isoform 1 precursor [Homo sapiens]

[0242]

[0243] "Fas cell surface death receptor (FAS) polynucleotide" means a nucleic acid encoding an FAS polypeptide. Exemplary FAS nucleic acid sequences are provided below.

[0244] >NM_000043.6 Homo sapiens Fas cell surface death receptor (FAS), transcript variant 1, mRNA

[0245]

[0246]

[0247]

[0248]

[0249] "Fragment" means a portion of a polypeptide or nucleic acid molecule. This portion contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. Fragments 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.

[0250] "Autologous killing" means that immune cells are killed by other immune cells, including immune cell killing driven by self-antigens. In certain embodiments, the immune cells of the present invention are genetically modified to prevent or reduce the expression of antigens recognized by immune cells expressing a chimeric antigen receptor (CAR), thereby preventing or reducing autologous killing. In various embodiments, autologous killing can occur in vivo (e.g., in a subject) or ex vivo (e.g., in an immune cell preparation).

[0251] "Graft-versus-host disease" (GVHD) refers to a pathological condition in which transplanted donor cells generate an immune response against host cells.

[0252] "Guide RNA" or "gRNA" means a polynucleotide that may be specific for a target sequence and can form a complex with a polynucleotide programmable nuclease domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is 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 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 contains two domains: (1) a domain that shares homology with the target nucleic acid (e.g., and guides binding of the Cas9 complex to the target); and (2) a domain that binds the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence called tracrRNA and contains 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 content of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including 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 content of each patent being incorporated herein by reference in its entirety. In some embodiments, the gRNA contains two or more domains (1) and (2) and can be an "extended gRNA". The extended gRNA will bind two or more Cas9 proteins and bind the target nucleic acid at two or more different regions, as described herein. The gRNA contains a nucleotide sequence complementary to the target site, which mediates binding of the 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 replaces thymine during DNA transcription.

[0253] "Heterodimer" means a fusion protein that contains two domains, such as a wild-type TadA domain and a variant of the 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).

[0254] "Host-versus-graft disease" (HVGD) refers to a pathological condition in which the host's immune system generates an immune response against the transplanted donor cells.

[0255] "Hybridization" means the 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.

[0256] "Immune cell" means a cell of the immune system that is capable of generating an immune response.

[0257] "Immune effector cell" means 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 (Th1), a T helper 2 (Th2) cell, or a CD4-expressing helper T cell (CD4+ T cell).

[0258] "Immune response regulatory gene" or "immune response regulator" means a gene that encodes a polypeptide involved in the regulation of an immune response. Immune response regulatory genes can regulate the immune response by multiple mechanisms or at different levels. For example, an immune response regulatory gene can inhibit or promote the activation of immune cells such as T cells. An immune response regulatory gene can increase or decrease the activation threshold of immune cells. In some embodiments, the immune response regulatory gene positively regulates an immune cell signal transduction pathway. In some embodiments, the immune response regulatory gene negatively regulates an immune cell signal transduction pathway. In some embodiments, the immune response regulatory gene encodes an antigen, an antibody, a cytokine, or a neuroendocrine.

[0259] "Immunogenic gene" means a gene that encodes a polypeptide capable of eliciting an immune response. For example, an immunogenic gene can encode an immunogen that elicits an immune response. In some embodiments, the immunogenic gene encodes a cell surface protein. In some embodiments, the immunogenic gene encodes a cell surface antigen or a cell surface marker. In some embodiments, the cell surface marker is a T cell marker or a B cell marker. In some embodiments, the immunogenic gene encodes 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.

[0260] 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 clip 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 EndoV 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 EndoV or a catalytically inactive hAAG.

[0261] In some embodiments, the base excision repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein that can inhibit the uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain contains wild-type UGI or a fragment of 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 excision repair inhibitor is an inhibitor of inosine base excision repair. In some embodiments, the base excision repair inhibitor is a "catalytically inactive inosine-specific nuclease" or a "dead inosine-specific nuclease". Without being bound by a particular theory, a catalytically inactive inosine glycosylase (e.g., alkyladenine glycosylase (AAG)) can bind inosine but cannot create an abasic site or remove the inosine, thereby spatially blocking the newly formed inosine moiety from the DNA damage / repair machinery. In some embodiments, the catalytically inactive inosine-specific nuclease may be able to bind inosine in a nucleic acid but not cleave the nucleic acid. Non-limiting exemplary catalytically inactive inosine-specific nucleases include catalytically inactive alkyladenosine glycosylase (AAG nuclease), e.g., from humans; and catalytically inactive endonuclease V (EndoV nuclease), e.g., from Escherichia coli. In some embodiments, the catalytically inactive AAG nuclease contains an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0262] "Increase" means a positive change of at least 10%, 25%, 50%, 75% or 100%.

[0263] An "intein" is a fragment of a protein that can excise itself during a process called protein splicing and join the remaining fragments (exteins) with a peptide bond. Inteins are also referred to as "protein introns". The process by which an intein excises itself and joins the remaining part of the 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. Herein, such an intein is referred to 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). Herein, the intein encoded by the dnaE-n gene may be referred to as "intein-N". Herein, the intein encoded by the dnaE-c gene may be referred to as "intein-C".

[0264] Other intein systems can also be used. For example, synthetic inteins based on the dnaE intein, namely Cfa-N (e.g., split-intein-N) and Cfa-C (e.g., split-intein-C) intein pairs 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 according to 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).

[0265] Exemplary nucleotide and amino acid sequences of inteins are provided below.

[0266] DnaE intein-N DNA:

[0267] TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTCTGCCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGGGGAGAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTTCCTAAT

[0268] DnaE intein-N protein:

[0269] CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN

[0270] DnaE intein-C DNA:

[0271] ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGATATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT

[0272] Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN

[0273] Cfa-N DNA:

[0274] TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCACAATCGCGGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA

[0275] Cfa-N protein:

[0276] CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP

[0277] Cfa-C DNA:

[0278] ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAAGTCTTGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC

[0279] Cfa-C protein: MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

[0280] 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 a structure of 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 a structure of N--[Intein-C]-[C-terminal portion of split Cas9]--C. The intein-mediated protein splicing mechanism for joining the protein to which the intein is fused (e.g., split Cas9) is known in the art, for example, as described in Shah et al., ChemSci. 2014; 5(1):446-461, which is incorporated herein by reference. Methods for designing and using inteins are known in the art and are described, for example, in WO2014004336, WO2017132580, US20150344549, and US20180127780, each of which is incorporated herein by reference in its entirety.

[0281] The terms "isolated", "purified", or "biologically pure" refer to materials that are, to varying degrees, free of the components that normally accompany them as seen in their native state. "Isolated" represents the degree of separation from the original source or surrounding material. "Purified" represents a degree of separation higher than 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 at the material level or cause other negative consequences. In other words, when produced by recombinant DNA techniques, a nucleic acid or peptide of the present invention 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 and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can indicate that a nucleic acid or protein gives rise to substantially a single band on an electrophoretic gel. For proteins that can be modified such as phosphorylated or glycosylated, different modifications can result in different isolated proteins, which can be purified independently.

[0282] "Isolated polynucleotide" means a nucleic acid (e.g., DNA) that does not contain genes that flank the gene in the natural genome of the organism from which the nucleic acid molecule of the present invention is derived. Thus, the term includes, for example, recombinant DNA incorporated into a vector, an autonomously replicating plasmid or virus, or incorporated into the genomic DNA of a prokaryote or eukaryote; or existing as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion). In addition, the term includes RNA molecules transcribed from DNA molecules, and recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0283] "Isolated polypeptide" means a polypeptide of the present invention that has been separated from its natural associated components. Typically, a polypeptide is free of at least 60% by weight of the proteins and natural organic molecules that are naturally associated with it. 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 present invention. The isolated polypeptides of the present invention can be obtained, for example, by extraction from natural sources, by expression of recombinant nucleic acids encoding such polypeptides, or by chemical synthesis of the protein. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0284] "Leader peptide" means a short amino acid sequence (e.g., about 16 to 30 amino acids in length) that directs a newly synthesized secreted or membrane protein to and across a membrane (e.g., the endoplasmic reticulum membrane). Leader peptides are typically located 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 CARs of the present invention include a leader peptide sequence (e.g., at the N-terminus of the antigen-binding domain). An exemplary leader peptide amino acid sequence is: METDTLLLWVLLLWVPGSTG.

[0285] 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, such as two components of a protein complex or ribonucleic acid complex, or two domains of a fusion protein, such as a polynucleotide programmable DNA binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine deaminase, cytidine deaminase). The linker can join different components of the base editor system or different parts of multiple components. For example, in some embodiments, the linker can join the guide polynucleotide binding domain of a polynucleotide programmable nucleotide binding domain to the catalytic domain of a deaminase. In some embodiments, the linker can join a CRISPR polypeptide to a deaminase. In some embodiments, the linker can join Cas9 to a deaminase. In some embodiments, the linker can join dCas9 to a deaminase. In some embodiments, the linker can join nCas9 to a deaminase. In some embodiments, the linker can join a guide polynucleotide to a deaminase. In some embodiments, the linker can join the deaminating component of the base editor system to the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can join the RNA binding portion of the deaminating component of the base editor system to the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can join the RNA binding portion of the deaminating component of the base editor system to the RNA binding portion of the polynucleotide programmable nucleotide binding component. The linker can be located between two groups, molecules, or other moieties or flanked by two groups, molecules, or other moieties and joined to each other via covalent bonds or non-covalent interactions, thereby joining the two. In some embodiments, the linker can be an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, the linker can comprise an aptamer capable of binding to a ligand. In some embodiments, the ligand can be a carbohydrate, peptide, protein, or nucleic acid. In some embodiments, the linker can comprise an aptamer that can be derived from a riboswitch. The riboswitch from which the aptamer is derived can be selected from the theophylline riboswitch, thiamine pyrophosphate (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, the 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, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile 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 nucleobase editing component can comprise a deaminase domain and an RNA recognition motif.

[0286] In some embodiments, the linker can be one amino acid or multiple amino acids (e.g., a peptide or 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.

[0287] 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 between two groups, molecules, or other moieties or flanks two groups, molecules, or other moieties and is covalently linked to each other to join the two. In some embodiments, the linker is one amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the 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, the linker comprises the amino acid sequence SGSETPGTSESATPES, also referred to as the XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker comprises an (SGGS)n, (GGGS)n, (GGGGS)n, (G)n, (EAAAK)n, (GGS)n, SGSETPGTSESATPES, or (XP)n motif, or any combination thereof, 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, the linker comprises multiple proline residues and is 5 to 21, 5 to 14, 5 to 9, or 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 "rigid" linkers.

[0288] In some embodiments, a 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 the 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.

[0289] In some embodiments, the domains of the base editor are fused via a linker that comprises the following amino acid sequences:

[0290] SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS.

[0291] In some embodiments, the domains of the base editor are fused via a linker that comprises the amino acid sequence SGSETPGTSESATPES, which may also be referred to as the 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.

[0292] 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, leukemia, myeloma, and liquid lymphoma. 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 regions. "Liquid cancer" can be recurrent, refractory, or metastatic. The liquid cancer to be treated using the methods described herein can be, for example, hairy cell lymphoma; liquid lymphomas include lymphomas that contain cysts or liquid regions.

[0293] "Lymphocyte activation gene 3 (LAG-3) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI accession number NP_002277.4 or a fragment thereof and has immunomodulatory activity. Exemplary amino acid sequences are provided below.

[0294] >NP_002277.4 Lymphocyte activation gene 3 protein precursor [Homo sapiens]

[0295]

[0296] "Lymphocyte activation gene 3 (LAG-3) polynucleotide" means a nucleic acid encoding an LAG-3 polypeptide. Exemplary LAG-3 nucleic acid sequences are provided below.

[0297] >NM_002286.6 Homo sapiens lymphocyte activation 3 (LAG3), mRNA

[0298]

[0299]

[0300] "Marker" means any protein or polynucleotide that has an alteration in expression level or activity that is associated with a disease or lesion.

[0301] As used herein, the term "mutation" refers to the replacement of a residue within a sequence (e.g., a nucleic acid or amino acid sequence) with another residue, or the deletion or insertion of one or more residues within the sequence. In the present context, a mutation is typically described as: the identity of the original residue, then the position of that residue within the sequence, and then the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art and are provided, for example, by 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 effectively generate "desired mutations" such as point mutations in nucleic acids (e.g., nucleic acids within a subject's genome) without generating a large 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 and that binds to a guide polynucleotide (e.g., a gRNA).

[0302] Typically, mutations made in or identified in a sequence (e.g., an amino acid sequence as described herein) are numbered relative to a reference (or wild-type) sequence (i.e., a sequence that does not contain the mutation). Those skilled in the art will readily understand how to determine the position of mutations in amino acid and nucleic acid sequences relative to a reference sequence.

[0303] "Tumorigenesis" refers to cells or tissues that exhibit abnormal growth or proliferation. The term tumorigenesis encompasses cancer, liquid and solid tumors. In some embodiments, tumorigenesis is a solid tumor. In other embodiments, tumorigenesis is a liquid tumor. In some embodiments, tumorigenesis is a hematological cancer. In some embodiments, the hematological cancer is leukemia, myeloma, and / or lymphoma. In some embodiments, the hematological cancer is a B-cell cancer. In some embodiments, the B-cell cancer is lymphoma or leukemia. In some cases, leukemia includes preleukemia. In some cases, leukemia is acute leukemia. Acute leukemia includes, for example, acute myeloid leukemia (AML). Acute leukemia also includes, for example, acute lymphoblastic leukemia or acute lymphocytic leukemia (ALL); ALL includes B-lineage ALL, T-lineage ALL, and T-cell acute lymphoblastic leukemia (T-ALL).

[0304] Non-limiting examples of tumorigenesis include 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 disease, extranodal NK / T-cell lymphoma, adult T-cell leukemia / lymphoma, T-cell prolymphocytic leukemia, subcutaneous panniculitis-like T-cell lymphoma, primary cutaneous γδ T-cell lymphoma, aggressive NK-cell leukemia, and enteropathy-associated T-cell lymphoma. In some embodiments, tumorigenesis is T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, tumorigenesis is T-cell acute myeloid leukemia (AML).

[0305] "Nuclear factor of activated T cells 1 (NFATc1) polypeptide" means a protein that has at least about 85% sequence identity with NCBI accession number NM_172390.2 or a fragment thereof and is a component of the activated T-cell DNA-binding transcriptional complex. Exemplary amino acid sequences are provided below.

[0306] >NP_765978.1 Nuclear factor of activated T cells, cytoplasmic 1 isoform A [Homo sapiens]

[0307] MPSTSFPVPSKFPLGPAAAVFGRGETLGPAPRAGGTMKSAEEEHYGYASSNVSPALPLPTAHSTLPAPCHNLQTSTPGIIPPADHPSGYGAALDGGPAGYFLSSGHTRPDGAPALESPRIEITSCLGLYHNNNQFFHDVEVEDVLPSSKRSPSTATLSLPSLEAYRDPSCLSPASSLSSRSCNSEASSYESNYSYPYASPQTSPWQSPCVSPKTTDPEEGFPRGLGACTLLGSPRHSPSTSPRASVTEESWLGARSSRPASPCNKRKYSLNGRQPPYSPHHSPTPSPHGSPRVSVTDDSWLGNTTQYTSSAIVAAINALTTDSSLDLGDGVPVKSRKTTLEQPPSVALKVEPVGEDLGSPPPPADFAPEDYSSFQHIRKGGFCDQYLAVPQHPYQWAKPKPLSPTSYMSPTLPALDWQLPSHSGPYELRIEVQPKSHHRAHYETEGSRGAVKASAGGHPIVQLHGYLENEPLMLQLFIGTADDRLLRPHAFYQVHRITGKTVSTTSHEAILSNTKVLEIPLLPENSMRAVIDCAGILKLRNSDIELRKGETDIGRKNTRVRLVFRVHVPQPSGRTLSLQVASNPIECSQRSAQELPLVEKQSTDSYPVVGGKKMVLSGHNFLQDSKVIFVEKAPDGHHVWEMEAKTDRDLCKPNSLVVEIPPFRNQRITSPVHVSFYVCNGKRKRSQYQRFTYLPANGNAIFLTVSREHERVGCFF

[0308] "Nuclear factor of activated T cells 1 (NFATc1) polynucleotide" means a nucleic acid encoding an NFATc1 polypeptide. The NFATc1 gene encodes a protein that is involved in the inducible expression of cytokine genes, especially IL-2 and IL-4, in T cells. Exemplary nucleic acid sequences are provided below.

[0309] >NM_172390.2 Homo sapiens Nuclear factor of activated T cells 1 (NFATC1), transcript variant 1, mRNA

[0310]

[0311] The term "non-conservative mutation" includes amino acid substitutions between different groups, for example, lysine replacing tryptophan, or phenylalanine replacing serine, etc. In such cases, 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.

[0312] The term "nuclear localization sequence", "nuclear localization signal", or "NLS" refers to an amino acid sequence that promotes the entry of a protein into the nucleus. Nuclear localization sequences are known in the art and are described, for example, in the international PCT application PCT / EP2000 / 011690 by Plank et al. filed on November 23, 2000 and published as WO / 2001 / 038547 on May 31, 2001, the content of which is incorporated herein by reference to its disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS described, for example, by Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequences PKKKRKVEGADKRTADGSEFESPKKKRKV, KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0313] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound that includes a nucleobase and an acidic moiety, e.g., a nucleoside, nucleotide, or polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules that include three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other via phosphodiester linkages. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., nucleotide and / or nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain that includes three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably and 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-stranded and / or double-stranded DNA. Nucleic acids can be naturally occurring, e.g., in the context of transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, cosmids, chromosomes, chromatids, or other naturally occurring nucleic acid molecules in a genome. On the other hand, nucleic acid molecules can be non-naturally occurring molecules, e.g., recombinant DNA or RNA, artificial chromosomes, engineered genomes, or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or include non-naturally occurring nucleotides or nucleosides. Additionally, the terms "nucleic acid", "DNA", "RNA", and / or similar terms include nucleic acid analogs, e.g., analogs having a backbone other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where applicable, e.g., in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs such as analogs having chemically modified bases or sugars, as well as backbone modifications. Unless otherwise specified, nucleic acid sequences are presented in the 5' to 3' direction. In some embodiments, a nucleic acid is or includes a natural nucleoside (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanosine, and 2-mercaptocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluoro-ribose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates 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 that 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., nuclease-active Cas9, Cas9 nickase (nCas9), or 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 / CasΦ.Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Cas12j / CasΦ, 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, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, 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, their homologs, or their modified or engineered versions. Other nucleic acid programmable DNA-binding proteins are also within the scope of the present disclosure, but they may not be specifically listed in the present disclosure. See, for example, 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 are incorporated herein by reference.

[0314] As used interchangeably herein, the terms "nucleobase", "nitrogenous base", or "base" refer to nitrogen-containing biological compounds that form nucleosides, which in turn are components of nucleotides. The ability of nucleobases to form base pairs and stack on top of one another directly results in long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleobases, namely adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), are referred to as primary or canonical nucleobases. Adenine and guanine are derived from purine, while cytosine, uracil, and thymine are derived from pyrimidine. DNA and RNA may also contain other (non-primary) bases that are modified. Non-limiting exemplary modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydroxymethylcytosine. Both hypoxanthine and xanthine can be generated by deamination (replacement of an amino group with a carbonyl group) in the presence of a mutagen. Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can result from the deamination 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.

[0315] 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-template 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 an engineered or evolved nucleobase editing domain from a naturally occurring nucleobase editing domain. The nucleobase editing domain can be from any organism, such as bacteria, human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse.

[0316] 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 as having, being predisposed to, or being at risk of developing a disease or disorder. In some embodiments, the term "patient" refers to a mammalian subject having a higher than average likelihood of developing a disease or disorder. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cows, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, gerbils, or guinea pigs), and other mammals that may benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female.

[0317] As used herein, "a patient in need thereof" or "a subject in need thereof" refers to a patient diagnosed as having, having, expected to have, or being predisposed to a disease or disorder (e.g., a T or NK cell malignancy) or being at risk of such disease or disorder.

[0318] The terms "disease-causing mutation", "disease-causing variant", "mutation causing disease", "variant causing disease", "harmful mutation", or "mutagenic mutation" refer to a genetic alteration or mutation that increases an individual's susceptibility or predisposition to a particular disease or disorder. In some embodiments, a disease-causing mutation comprises at least one wild-type amino acid substitution by at least one disease-causing amino acid in a protein encoded by the gene.

[0319] 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, magnesium stearate, calcium stearate, or zinc stearate, or stearic acid), or solvent encapsulating material involved in carrying or transporting a compound from one site in the body (e.g., the delivery site) to another site (e.g., an organ, tissue, or part of the body). A pharmaceutically acceptable carrier is "acceptable" in that it is compatible with the other ingredients of the formulation and does not injure the tissues of the subject (e.g., physiologically compatible, sterile, physiological pH, etc.). Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier", "vehicle", etc. are used interchangeably herein.

[0320] The term "pharmaceutical composition" means a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional reagents (e.g., for specific delivery, increasing half-life, or other therapeutic compounds).

[0321] "Programmed cell death 1 (PDCD1 or PD-1) polypeptide" means a protein having at least about 85% amino acid sequence identity with NCBI accession number AJS10360.1 or a fragment thereof. The PD-1 protein is thought to be involved in the regulation of T cell function during an immune response and in tolerance conditions. Exemplary B2M polypeptide sequences are provided below.

[0322] >AJS10360.1 Programmed cell death 1 protein [Homo sapiens] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0323] "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 inhibits T cell effector function in an antigen-specific manner. Exemplary PDCD1 nucleic acid sequences are provided below.

[0324] >AY238517.1 Homo sapiens Programmed cell death 1 (PDCD1) mRNA, complete cds

[0325] ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTCTGA

[0326] The terms "protein", "peptide", "polypeptide" and their grammatical equivalents are used interchangeably herein and refer to polymers 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, the length of a protein, peptide or polypeptide will be at least three amino acids. A protein, peptide or polypeptide can refer to a single protein or a collection of proteins. One or more of the amino acids in a protein, peptide or polypeptide can be modified, for example, by the addition of chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl, geranylgeranyl, fatty acid groups; linkers for conjugation; functionalization; or other modifications, etc. A protein, peptide or polypeptide can also be a single molecule or can be a multimolecular 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 contains protein domains from at least two different proteins. A protein can be located in the amino-terminal (N-terminal) portion of the fusion protein or at the carboxyl-terminal (C-terminal) protein, thus forming an amino-terminal fusion protein or a carboxyl-terminal fusion protein, respectively. A protein can contain different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9, which guides the binding of the protein to a target site) and a nucleic acid cleavage domain, or the catalytic domain of a nucleic acid editing protein. In some embodiments, a protein contains a protein portion (e.g., the 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 protein 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 containing peptide linkers. Methods for recombinant protein expression and purification are well known and include those described in Molecular Cloning: A Laboratory Manual (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).

[0327] The polypeptides and proteins (including their functional portions and functional variants) disclosed herein may contain 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, aminocyclohexanecarboxylic acid, norleucine, α-aminodecanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-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, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norleucine)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, norvaline, and α-tert-butylglycine. The polypeptides and proteins may 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 ethylization), ubiquitination, addition of pyrrolidonecarboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylgeranylation, glycosylphosphatidylinositolization, lipidation, and iodination.

[0328] "Promoter" means an array of nucleic acid control sequences that direct the transcription of a nucleic acid. The promoter includes the essential nucleic acid sequences proximal to the transcription start site. The promoter also optionally includes distal enhancer or silencer sequence elements. A "constitutive promoter" is a promoter that is continuously active and not regulated by external signals or molecules. In contrast, the activity of an "inducible promoter" is regulated by external signals or molecules (e.g., transcription factors). By way of example, the promoter can be the CMV promoter.

[0329] 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 the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule contains an amino acid or nucleotide sequence that contains 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.

[0330] "Reduce" means a reverse change of at least 10%, 25%, 50%, 75%, or 100%.

[0331] "Reference" means a standard or control condition. In one embodiment, the reference can be a wild-type or healthy cell. In other embodiments and without limitation, the reference is a cell that has not been treated, a cell that has not been subjected to the test conditions or has been subjected to a placebo or saline, vehicle, buffer, and / or a control vector that does not carry the polynucleotide of interest.

[0332] "Reference sequence" is a defined sequence used as a basis for sequence alignment. The reference sequence can be a subset or the entirety of a particular sequence, e.g., a segment of a full-length cDNA or gene sequence, or a 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 integer number of nucleotides thereabout or therebetween. In some embodiments, the reference sequence is the wild-type sequence of the protein of interest. In other embodiments, the reference sequence is the polynucleotide sequence encoding the wild-type protein.

[0333] The terms “RNA programmable nuclease” and “RNA-guided nuclease” are used in conjunction with (e.g., bound or associated with) one or more RNAs that are not the cleavage target. In some embodiments, when an RNA programmable nuclease is complexed with an RNA, it may be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). The gRNA may exist as a complex of two or more RNAs, or as a single RNA molecule. A gRNA that exists as a single RNA molecule may 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 contains two domains: (1) a domain that shares homology with the target nucleic acid (e.g., and guides binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence called tracrRNA and contains 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 content of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. Patent Application No. 61 / 874,682, entitled “Switchable Cas9 Nucleases and Uses Thereof,” filed Sep. 6, 2013, and U.S. Patent Provisional Application No. 61 / 874,746, entitled “Delivery System For Functional Nucleases,” filed Sep. 6, 2013, the entire contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the gRNA contains two or more domains (1) and (2) and can be an “extended gRNA.” For example, an extended gRNA will bind two or more Cas9 proteins and bind the target nucleic acid at two or more different regions, as described herein. The gRNA contains a nucleotide sequence complementary to the target site, which mediates binding of the nuclease / RNA complex to the target site, providing sequence specificity of the nuclease:RNA complex.

[0334] In some embodiments, the RNA-programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 (Csn1) from Streptococcus pyogenes (see, e.g., “Complete genome sequence of an M1 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 DNA cleavage sites, these proteins can in principle be targeted to any sequence specific to the guide RNA.Methods for site-specific cleavage (e.g., to modify the genome) using RNA-programmable nucleobases such as Cas9 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 ah RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013); the respective entire contents of which are incorporated herein by reference).

[0335] "Signaling domain" means the intracellular portion of a protein expressed within a T cell that transduces signals for T cell effector functions (e.g., activation signals) and directs the T cell to perform specialized functions. T cell activation can be induced by a variety of factors, including the binding of antigen to the T cell receptor on the surface of the T cell and the binding of a cognate ligand to a co-stimulatory molecule on the surface of the T cell. T cell co-stimulatory molecules are cognate binding partners on T cells that specifically bind to co-stimulatory ligands, thereby mediating co-stimulatory responses of the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to, MHC class I molecules. In some embodiments, the co-stimulatory domain is the CD2 cytoplasmic domain. Activation of the T cell results in 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 the CD2, CD3ζ, CD8, CD28, CD27, CD154, GITR (TNFRSF18), CD134 (OX40), and CD137 (4-1BB) signaling domains.

[0336] "Single-chain antibody" or "scFv" means a genetically engineered molecule that contains the VH and VL domains of one or more antibodies linked as a single-chain molecule by a verified peptide linker (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 intra-molecular orientation of the VH and VL domains in the scFv is VH domain-linker domain-VL domain. In some embodiments, the intra-molecular orientation of the VH and VL domains in the scFv is VL domain-linker domain-VH domain.

[0337] 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, the C nucleotide may be present in most individuals, but in a few individuals, this position is occupied by an A. This means that an SNP exists at this specific location, and the two possible nucleotide variations, C or A, are called the alleles at this position. SNPs are the basis for differences in disease susceptibility. The severity of a disease and our body's response to treatment are also manifestations of genetic variations. SNPs can fall within the coding region of a gene, within the non-coding region of a gene, or within the intergenic region (the region between genes). In one embodiment, due to the degeneracy of the genetic code, an SNP within a coding sequence does not necessarily change the amino acid sequence of the resulting protein. There are two types of SNPs within the coding region: synonymous and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs change the amino acid sequence of the protein. Non-synonymous SNPs are of two types: missense and nonsense. SNPs that are not within the protein-coding region can still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNAs. Gene expression affected by this type of SNP is called eSNP (expressed SNP) and can be upstream or downstream of the gene. A single nucleotide variant (SNV) is a variation 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 called single nucleotide alterations.

[0338] "Specifically binds" means that a nucleic acid molecule, polypeptide, or a 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 the polypeptide and / or nucleic acid molecule of the present invention, but substantially does not recognize and bind to other molecules in a sample (e.g., a biological sample). For example, a chimeric antigen receptor specifically binds to a specific marker expressed on the cell surface, but does not bind to other polypeptides, carbohydrates, lipids, or any other compound on the cell surface.

[0339] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention 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. Polynucleotides having "substantial identity" to an endogenous sequence will typically hybridize to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention 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. Polynucleotides having "substantial identity" to an endogenous sequence will typically hybridize to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" means the pairing between complementary polynucleotide sequences (e.g., the genes described herein) or fragments thereof to form double-stranded molecules under various stringent conditions. (See, e.g., Wahl, G.M. and S.L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

[0340] For example, stringent salt concentrations will generally be less than about 750 mM NaCl and 75 mM sodium citrate, preferably less than 500 mM NaCl and 50 mM sodium citrate, and more preferably about 250 mM NaCl and 25 mM sodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents such as formamide, while high stringency hybridization can be 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 surfactants such as sodium dodecyl sulfate (SDS), and inclusion or non-inclusion of carrier DNA are well known to those skilled in the art. By combining these conditions as needed, various levels of stringency can be achieved. In one embodiment, hybridization will occur at 30°C in 750 mM NaCl, 75 mM sodium citrate, and 1% SDS. In another embodiment, hybridization will occur at 37°C in 500 mM NaCl, 50 mM sodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In another embodiment, hybridization will occur at 42°C in 250 mM NaCl, 25 mM sodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. The available variations of these conditions will be apparent to those skilled in the art.

[0341] For most applications, the wash step after hybridization will also alter the stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As noted above, wash stringency can be increased by decreasing the salt concentration or by increasing the temperature. For example, a stringent salt concentration for a wash 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. Stringent temperature conditions for a wash step will generally include a temperature of at least about 25° C., more preferably at least about 42° C., and even more preferably at least about 68° C. In one embodiment, the wash step will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash 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 wash 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, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Current Protocols in Molecular Biology (Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001)); Guide to Molecular Cloning Techniques (Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York)); and Molecular Cloning: A Laboratory Manual (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York).

[0342] "Split" means divided into two or more segments.

[0343] "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 and C-terminal portions of the Cas9 protein can be spliced to form a "reconstituted" Cas9 protein. In certain embodiments, the Cas9 protein is split into two fragments located within the disordered regions of the protein, e.g., 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 at any C, T, A, or S within the regions located between amino acids A292 to G364, F445 to K483, or E565 to T637 of SpCas9 or at the corresponding positions within any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In some embodiments, the protein is split into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of splitting the protein into two fragments is referred to as "splitting" the protein.

[0344] In other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1 to 573 or 1 to 637 of Streptococcus 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 a portion of amino acids 574 to 1368 or 638 to 1368 of SpCas9 wild-type or the corresponding positions thereof.

[0345] The C-terminal portion of split Cas9 can be joined to 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 amino acids (551 to 651) to 1368 of spCas9. "(551 to 651) to 1368" means starting at the amino acid between amino acids 551 to 651 (inclusive) and ending at amino acid 1368.For example, the C-terminal portion of split Cas9 may comprise any one 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 the split Cas9 protein comprises a portion of amino acids 574 to 1368 or 638 to 1368 of SpCas9.

[0346] "Subject" means a mammal, including but not limited to, humans and non-human mammals such as cows, horses, dogs, sheep or cats. Subjects include domestic animals, domesticated animals used for providing labor and providing daily necessities such as food, including but not limited to, cows, goats, chickens, horses, pigs, rabbits and sheep.

[0347] "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 being compared.

[0348] Typically, sequence analysis software is used (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) to measure sequence identity. Such software matches identical or similar sequences by assigning a degree of homology to different substitutions, deletions, and / or other modifications. Conservative substitutions typically include within-group substitutions within each of the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, and a probability score between e-3 and e-100 indicates closely related sequences.

[0349] Using COBALT, for example, the following parameters are employed:

[0350] a) Alignment parameters: gap penalty -11, -1, and end gap penalty -5, -1,

[0351] b) CDD parameters: Open with RPS BLAST; Blast E value 0.003; Find Conserved columns and Recompute are open, and

[0352] c) Query clustering parameters: Open with query clusters; word length 4; maximum inter-cluster distance 0.8; AlphabetRegular.

[0353] Use EMBOSS Needle, for example, with the following parameters:

[0354] a) Matrix: BLOSUM62;

[0355] b) GAP OPEN: 10;

[0356] c) GAP EXTEND: 0.5;

[0357] d) OUTPUT FORMAT; pair;

[0358] e) END GAP PENALTY: false;

[0359] f) END GAP OPEN: 10; and

[0360] g) END GAP EXTEND: 0.5.

[0361] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a base editor. In one embodiment, the target site is determined by a deaminase or a fusion protein comprising a deaminase (e.g., cytidine or adenosine deaminase).

[0362] "T cell receptor alpha constant region (TRAC) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI accession number P01848.2 or a fragment thereof and has immunomodulatory activity. Exemplary amino acid sequences are provided below.

[0363] >sp|P01848.2|TRAC_HUMAN RecName: Full=T cell receptor alpha constant region IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0364] "T cell receptor alpha constant region (TRAC) polynucleotide" means a nucleic acid encoding a TRAC polypeptide. Exemplary TRAC nucleic acid sequences are provided below.

[0365] UCSC Human Genome Database, gene ENSG00000277734.8 human T cell receptor alpha chain (TCR-α)

[0366]

[0367] The nucleotides represented by lowercase letters in the above text are untranslated regions or introns, and the nucleotides represented by uppercase letters are exons.

[0368] >X02592.1 mRNA of human T cell alpha chain (TCR-alpha)

[0369]

[0370] "T cell receptor beta constant region 1 polypeptide (TRBC1)" means a protein that has at least about 85% amino acid sequence identity with NCBI accession number P01850 or a fragment thereof and has immunomodulatory activity. Exemplary amino acid sequences are provided below.

[0371] >sp|P01850|TRBC1_HUMAN T cell receptor beta constant region 1 OS=Homo sapiens OX=9606 GN=TRBC1 PE=1 SV=4

[0372] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

[0373] "T cell receptor beta constant region 1 polynucleotide (TRBC1)" means a nucleic acid encoding the TRBC1 polypeptide. Exemplary TRBC1 nucleic acid sequences are provided below.

[0374] >X00437.1

[0375]

[0376] "T cell receptor beta constant region 2 polypeptide (TRBC2)" means a protein that has at least about 85% amino acid sequence identity with NCBI accession number A0A5B9 or a fragment thereof and has immunomodulatory activity. Exemplary amino acid sequences are provided below.

[0377] >sp|A0A5B9|TRBC2_HUMAN T cell receptor beta constant region 1 OS=Homo sapiens OX=9606 GN=TRBC2 PE=2 SV=2

[0378] DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0379] "T cell receptor beta constant region 2 polynucleotide (TRBC2)" means a nucleic acid encoding a TRAC polypeptide. Exemplary TRBC2 nucleic acid sequences are provided below.

[0380]

[0381] "tet methylcytosine dioxygenase 2 (TET2) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI accession number FM992369.1 or a fragment thereof and has catalytic activity for converting methylcytosine to 5-hydroxymethylcytosine. Defects in this gene have been associated with myeloproliferative disorders, and the enzyme's ability to methylate cytosine contributes to transcriptional regulation. Exemplary TET2 amino acid sequences are provided below.

[0382] >CAX30492.1 tet oncogene family member 2 [Homo sapiens]

[0383]

[0384] "tet methylcytosine dioxygenase 2 (TET2) polynucleotide" means a nucleic acid molecule encoding a TET2 polypeptide. The TET polypeptide encodes a methylcytosine dioxygenase and has transcriptional regulatory activity. Exemplary TET2 nucleic acid sequences are presented below.

[0385]

[0386] "Transforming growth factor receptor 2 (TGFBRII) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI accession number ABG65632.1 or a fragment thereof and has immunosuppressive activity. Exemplary amino acid sequences are provided below.

[0387] >ABG65632.1 Transforming growth factor beta receptor II [Homo sapiens] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

[0388] "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. Exemplary TGFBRII nucleic acid sequences are provided below.

[0389] >M85079.1 Human TGF-beta type II receptor mRNA, complete coding sequence

[0390]

[0391] "T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI accession number ACD74757.1 or a fragment thereof and has immunomodulatory activity. Exemplary TIGIT amino acid sequences are provided below.

[0392] >ACD74757.1 T cell immunoreceptor with Ig and ITIM domains [Homo sapiens]

[0393] MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG

[0394] "T cell immunoreceptor with Ig and ITIM domains (TIGIT) polynucleotide" means a nucleic acid encoding a TIGIT polypeptide. The TIGIT gene encodes an inhibitory immunoreceptor associated with tumorigenesis and T cell exhaustion. Exemplary nucleic acid sequences are provided below.

[0395] >EU675310.1 Homo sapiens T cell immunoreceptor with Ig and ITIM domains (TIGIT) mRNA, complete cds

[0396] CGTCCTATCTGCAGTCGGCTACTTTCAGTGGCAGAAGAGGCCACATCTGCTTCCTGTAGGCCCTCTGGGCAGAAGCATGCGCTGGTGTCTCCTCCTGATCTGGGCCCAGGGGCTGAGGCAGGCTCCCCTCGCCTCAGGAATGATGACAGGCACAATAGAAACAACGGGGAACATTTCTGCAGAGAAAGGTGGCTCTATCATCTTACAATGTCACCTCTCCTCCACCACGGCACAAGTGACCCAGGTCAACTGGGAGCAGCAGGACCAGCTTCTGGCCATTTGTAATGCTGACTTGGGGTGGCACATCTCCCCATCCTTCAAGGATCGAGTGGCCCCAGGTCCCGGCCTGGGCCTCACCCTCCAGTCGCTGACCGTGAACGATACAGGGGAGTACTTCTGCATCTATCACACCTACCCTGATGGGACGTACACTGGGAGAATCTTCCTGGAGGTCCTAGAAAGCTCAGTGGCTGAGCACGGTGCCAGGTTCCAGATTCCATTGCTTGGAGCCATGGCCGCGACGCTGGTGGTCATCTGCACAGCAGTCATCGTGGTGGTCGCGTTGACTAGAAAGAAGAAAGCCCTCAGAATCCATTCTGTGGAAGGTGACCTCAGGAGAAAATCAGCTGGACAGGAGGAATGGAGCCCCAGTGCTCCCTCACCCCCAGGAAGCTGTGTCCAGGCAGAAGCTGCACCTGCTGGGCTCTGTGGAGAGCAGCGGGGAGAGGACTGTGCCGAGCTGCATGACTACTTCAATGTCCTGAGTTACAGAAGCCTGGGTAACTGCAGCTTCTTCACAGAGACTGGTTAGCAACCAGAGGCATCTTCTGG

[0397] As used herein, "transduction" means the transfer of a gene or genetic material into a cell via a viral vector.

[0398] As used herein, "transformation" refers to the process of introducing a genetic change into a cell by the introduction of exogenous nucleic acid.

[0399] "Transfection" refers to the transfer of genes or genetic material into cells by chemical or physical means.

[0400] "Translocation" means the rearrangement of nucleic acid segments between non-homologous chromosomes.

[0401] "Transmembrane domain" means an amino acid sequence that inserts into a lipid bilayer, such as the lipid bilayer of a cell or a virus or virus-like particle. Transmembrane domains can be used to anchor a protein of interest (e.g., a CAR) to the membrane. Transmembrane domains can be derived from natural sources or from synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane domains for the disclosed CARs can include at least the transmembrane regions of the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, 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, CD8α, CD28, and CD3ζ. In some embodiments, the transmembrane domain is the CD8α hinge and transmembrane domain.

[0402] As used herein, the terms "treating", "treatment", etc. refer to alleviating or relieving a disease or disorder and / or its associated symptoms or obtaining a desired pharmacological and / or physiological effect. It should be understood that, although not excluded, treating a lesion or condition does not require complete elimination of the lesion, condition, or associated symptoms. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely alleviates, eliminates, abolishes, attenuates, slows, reduces the disease and / or the intensity of the negative symptoms attributable to the disease or cures the disease and / or the negative symptoms attributable to the disease. In some embodiments, the effect is prophylactic, i.e., the effect protects or prevents the occurrence or recurrence of a disease or disorder. To this end, the methods of the present disclosure include administering a therapeutically effective amount of the compositions described herein.

[0403] 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 a fragment thereof that binds to the host uracil-DNA glycosylase and prevents the removal of uracil residues from DNA. In one embodiment, UGI is a protein, a fragment or a domain thereof that is capable of inhibiting the uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or a modified version thereof. In some embodiments, the UGI domain comprises a fragment of the exemplary amino acid sequence detailed below. In some embodiments, the UGI fragment comprises an amino acid sequence that comprises 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 the exemplary UGI sequence provided below. In some embodiments, UGI comprises an amino acid sequence that is homologous to the exemplary UGI amino acid sequence or a fragment thereof detailed below. In one embodiment, UGI or a 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 the wild-type UGI or UGI sequence or a fragment thereof detailed below. The exemplary UGI comprises the following amino acid sequence:

[0404] >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor

[0405] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVM LLTSDAPEYKPWALVIQDSNGENKIKML. The term "vector" means a means for introducing a nucleic acid sequence into a cell to obtain a transformed cell. Vectors include plasmids, transposons, bacteriophages, viruses V liposomes and episomes. An "expression vector" is a nucleic acid sequence that contains a nucleic acid sequence to be expressed in a recipient cell. The expression vector may include additional nucleic acid sequences to facilitate and / or promote the expression of the introduced sequence such as initiators, terminators, promoters and secretion sequences.

[0406] "T cell receptor ζ-chain-associated protein kinase 70 (ZAP70) polypeptide" means a protein that has at least about 85% amino acid sequence identity with NCBI accession number AAH53878.1 and has kinase activity. The exemplary amino acid sequence is provided below.

[0407] >AAH53878.1 Zeta-chain (TCR) - associated protein kinase 70 kDa [Homo sapiens]

[0408] MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA

[0409] "T cell receptor zeta-chain associated protein kinase 70 (ZAP70)" means a nucleic acid encoding a ZAP70 polypeptide. The ZAP70 gene encodes a tyrosine kinase involved in T cell development and lymphocyte activation. Deletion of functional ZAP10 can result in severe combined immunodeficiency characterized by a lack of CD8+ T cells. Exemplary ZAP70 nucleic acid sequences are provided below.

[0410] >BC053878.1 Homo sapiens zeta-chain (TCR) - associated protein kinase 70 kDa, mRNA (cDNA clone MGC:61743 IMAGE:5757161), complete coding sequence

[0411]

[0412] Any 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 listed groups. Any recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0413] Any composition or method provided herein can be used in combination with one or more of any other compositions and methods provided herein.

[0414] DNA editing has become a viable means of altering disease states by correcting disease-causing mutations at the gene level. Until recently, all DNA editing platforms functioned by inducing DNA double-strand breaks (DSBs) at specific genomic loci and relying on endogenous DNA repair pathways to determine product outcomes in a semi-random manner, resulting in complex populations of genetic products. Although precise, user-defined repair outcomes can be achieved through the homology-directed repair (HDR) pathway, efficient repair using HDR poses many challenges in therapeutically relevant cell types. In practice, this pathway is inefficient compared to the competing, error-prone non-homologous end joining pathway. Moreover, HDR is strictly restricted to the G1 and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells. Thus, it has proven difficult or impossible to efficiently alter genomic sequences in a user-defined, programmable manner in these populations.

[0415] Incorporation by reference

[0416] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Brief Description of the Drawings

[0417] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention can be obtained by reference to the following detailed description of exemplary embodiments that utilize the principles of the invention, as well as the accompanying drawings:

[0418] Figures 1A to 1B is an illustrative illustration of three proteins that affect T cell function. Figure 1A is an illustrative illustration of the TRAC protein, which is a key component of graft-versus-host disease. Figure 1B is an illustrative illustration of the B2M protein, which is a component of the MHC class I antigen presentation complex present on nucleated cells and can be recognized by the host's CD8+ T cells. Figure 1CIs an illustrative description of T cell signaling that leads to the expression of the PDCD1 gene, and the resulting PD-1 protein functions to inhibit T cell signaling.

[0419] Figure 2 Is a graph of the percentage of cells with knockdown of target gene expression after base editing. "EP" indicates electroporation.

[0420] Figure 3 Is a graph of the percentage of types of gene modifications observed in untransduced cells or cells transduced with the BE4 base editing system or Cas9 nuclease.

[0421] Figure 4 Is a graph depicting the percentage of target nucleotide modifications measured by the percentage of cells in which target protein expression is negative as determined by flow cytometry (FC) in cells transduced with BE4 and sgRNAs that direct BE4 to the splice site acceptor (SA) or donor (SD), or cells that generate a STOP codon. Control cells were electroporated with a mock (EP).

[0422] Figure 5 Is a schematic diagram of the BE4 system disrupting the splice site acceptor (SA), splice donor (SD), or generating a STOP codon.

[0423] Figure 6 Is a chart summarizing the off-target binding sites of sgRNAs used to disrupt target genes.

[0424] Figure 7 Is a graph summarizing flow cytometry (FC) data of the percentage of cells edited with BE4 or Cas9 that exhibit reduced protein expression. Cells were gated to B2M or CD3, the latter being an indicator of TRAC expression.

[0425] 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.

[0426] Figure 9 Is a graph illustrating the effectiveness of the base editing techniques described herein in modifying specific genes that may have a negative impact on CAR-T immunotherapy.

[0427] Figure 10 Is a schematic diagram of a droplet digital PCR (ddPCR) protocol for detecting and quantifying gene modifications and translocations.

[0428] Figure 11Two figures are presented, showing data generated from next-generation sequencing (NGS) analysis or ddPCR of cells edited using the BE4 system or the Cas9 system.

[0429] Figure 12 It is a schematic diagram exemplifying the role played by Cbl-b in repressing T cell activation.

[0430] Figure 13 It is a figure depicting the efficiency of Cbl-b knockdown by disrupting the splice site. SA = splice acceptor; SD = splice donor; STOP – generates a STOP codon; 2°Only = secondary antibody only; C373 refers to the loss-of-function variant (C373R); RL1-A::APC-A = laser; ICS = intracellular staining.

[0431] Figure 14 It is a schematic diagram exemplifying the Cas12b-mediated indel rates in the GRIN2B and DNMT1 genes in T cells. EP represents electroporation.

[0432] Figure 15 It is a figure summarizing fluorescence-activated cell sorting (FACS) data of cells transduced by electroporation (EP) with bvCas12b and guide RNAs specific for TRAC, GRIN2B, and DNMT1 and gated against CD3.

[0433] Figure 16 It is a scatter plot of fluorescence-activated cell sorting data of cells transduced with CAR-P2A-mCherry lentivirus, indicating CAR expression.

[0434] Figure 17 It is a scatter plot of fluorescence-activated sorting data, indicating CAR expression in cells transduced with a poly(1,8-octanediol citrate) (POC) lentiviral vector.

[0435] Figure 18 It is a figure showing that BE4 produces effective and persistent gene knockout with high product purity.

[0436] Figure 19A It is a representative FACS analysis showing loss of protein surface expression due to gene knockout by BE4 or spCas9. Figure 19B It is a figure showing that gene knockout by BE4 or spCas9 results in loss of B2M surface expression.

[0437] Figure 20 It is a schematic diagram depicting the localization of B2M, TRAC, and PD-1 target sites. Translocations can be detected when the B2M, TRAC, and PD-1 sequences are recombined.

[0438] Figure 21 It is a figure showing that multiplex base editing does not significantly impair cell expansion.

[0439] Figure 22 It is a figure showing that BE4 generates triple-edited T cells with on-target editing efficiency and cell phenotypes similar to those of spCas9.

[0440] Figure 23 Describes flow cytometry analysis showing the generation of triple-edited CD3-, B2M-, PD1-T cells.

[0441] Figure 24 Describes flow cytometry analysis showing CAR expression in cells edited with BE4 and Cas9.

[0442] Figure 25 It is a figure showing that CAR-T cells kill antigen-positive cells.

[0443] Figure 26 It is a figure showing that Cas12b and BE4 can be paired for efficient multiplex editing in T cells.

[0444] Figure 27 It is a figure showing that Cas12b can direct the insertion of a chimeric antigen receptor (CAR) into a locus by introducing a double-stranded DNA template encoding the CAR into cells in the presence of a Cas12 nuclease and an sgRNA targeting the locus.

[0445] Figure 28A and Figure 28B It is an illustrative diagram showing base editing pathways used to silence genes for engineering CAR-T cells. Figure 28A Illustrates targets for CAR-T editing. Figure 28B Shows two strategies for silencing using base editors: creating a stop codon with a CBE and disrupting splicing with a CBE.

[0446] Figure 29 It is an illustrative cartoon showing that in some cases, multiple CAR-Ts are required to address clonality in AML.

[0447] Figure 30 It is an illustrative cartoon showing that in some cases, multiplex editing is required to eliminate fratricide in T-ALL CAR-T combinations.

[0448] Figure 31A and Figure 31B Illustrative description of the results of an experiment testing four simultaneous base edits for T-ALL; CAR-T does not affect yield compared to nucleases. Figure 31AExemplarily illustrate the theoretical yields in four-site T-ALL editing competitions for the following cases: 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 Exemplarily illustrate the cell viability after electroporation. For each set of results at 24 hours (hr), 48 hr, 72 hr, 96 hr, and 168 hr, the results are shown from right to left as no EP, EP only, CBE variant 1, CBE variant 2, and Cas9.

[0449] Figure 32 Exemplarily illustrate that more than 90% of the four-site knockouts were caused by BE4. Electroporation was performed at a scale of 5M cells, and quadruple editing was carried out in a single electroporation (EP) step. For all 4 targets, an editing efficiency greater than 90% was achieved using rBE4. For each set of results, the data are from left to right: PD1, CD7, TRAC, and CD52.

[0450] Figure 33 Exemplarily illustrate that base editing did not cause a difference in cell yields. The data are: no P (circles), EP only (squares), rBE4 (triangles), ppBE4 (inverted triangles), and Cas9 (diamonds).

[0451] Figure 34 Exemplarily illustrate CD3 and CD7 on edited CAR-T targeted model tumor cells. The data are: UTD 1:1 (circles), 7CAR8 1:1 (squares), and 3CAR2 1:1 (triangles).

[0452] Figure 35 Is a schematic diagram of an exemplary CD7 CAR-T cell for targeting T-AL tumor cells.

[0453] Figure 36 Is a flow chart describing the clinical protocol for treating patients with CD7 CAR-T cells.

[0454] Figures 37A to 37C Describe the production of CD7 CAR-T cells. Figure 37A Is a flow chart describing the protocol for producing TALL017 heterologous-fused CD7 CAR-T cells. Figure 37B Is a flow chart describing the protocol for producing TALL083 CD7 CAR-T cells. Figure 37C Is a scatter plot of fluorescence-activated cell sorting data indicating that TALL017 CAR-T cells are highly activated after fusion.

[0455] Figure 38It is a graph depicting the total number of edits in TALL017 and TALL038 CD7 CAR-T cells measured by next-generation sequencing (NGS).

[0456] Figure 39A and Figure 39B Describes the expression of TCRα / β, CD7, and CD52 in TALL017 and TALL038 CD7 CAR-T cells 24 hours after thawing. Figure 39A It is a scatter plot of fluorescence-activated cell sorting data for TALL017 and TALL038 CD7 CAR-T cells. Figure 39B It is a graph depicting the residual protein expression measured via FACS.

[0457] Figure 40 Describes the gating strategy for the identity panel.

[0458] Figure 41 It is a graph depicting the identity of the final cell product as a mixture of CD2+ / - and CD56+ / - cells.

[0459] Figure 42 It is a graph depicting the expression of CD7 CAR-T cells at 24 and 48 hours after thawing.

[0460] Figure 43 Includes multiple graphs that describe that the CD25 expression in TALL038 CD7 CAR-T cells is lower than that in TALL017 CD7 CAR-T cells after thawing.

[0461] Figure 44 It is a flow chart describing the potency protocol based on CD7 CAR-T beads for in vitro characterization.

[0462] Figure 45A and Figure 45B Describes that TALL038 CD7 CAR-T cells release IFNγ, TNFα, and IL-2 in response to the CD7 antigen. Figure 45A It is a graph depicting the release of IFNγ by TALL038 CD7 CAR-T cells. Figure 45B It is a graph depicting the release of TNFα, IL-10, and IL-2 by TALL038 CD7 CAR-T cells.

[0463] Figure 46 It is a schematic diagram describing the co-culture protocol for measuring CAR-directed T cell killing of tumor cells.

[0464] Figure 47A and Figure 47BDescription: Compared with TALL017 CAR T, TALL038 CD7 CAR-T cells showed increased CCRF upon rechallenge. Figure 47A It is a figure depicting the main challenge. Figure 47B It is a figure depicting the secondary challenge.

[0465] Figure 48 It is a figure depicting the bioluminescence emission data (mean, SEM) at day 10 post-CCRF transplantation / day -1 of CD7 CAR-T treatment. Total flux was measured on a linear scale.

[0466] Figure 49A and Figure 49B Describes the bioluminescence emission data (mean, SEM) at day 19 post-CCRF transplantation / day 8 post-CD7 CAR-T treatment. Figure 49A It is a figure depicting the average tumor burden. Total flux was measured on a linear scale. Figure 49B It is a figure depicting the average mouse weight.

[0467] Figure 50A and Figure 50B Describes the bioluminescence emission data (mean, SEM) at day 38 post-CCRF transplantation / day 27 post-CD7 CAR-T treatment. Figure 50A It is a figure depicting the average tumor burden. Total flux was measured on a linear scale. Figure 50B It is a figure depicting the average tumor burden. Total flux was measured on a logarithmic scale.

[0468] Figure 51A and Figure 51B Describes the bioluminescence emission data (individual mice) at day 38 post-CCRF transplantation / day 27 post-CD7 CAR-T treatment. Figure 51A It is a figure depicting the average tumor burden. Total flux was measured on a linear scale. Figure 51B It is a figure depicting the average tumor burden. Total flux was measured on a logarithmic scale.

[0469] Figure 52A and Figure 52B Describes the bioluminescence emission data (individual mice) at day 38 post-CCRF transplantation / day 27 post-CD7 CAR-T treatment. Figure 52A Includes figures depicting the average tumor burden. Total flux was measured on a linear scale (upper panel) and a logarithmic scale (lower panel). Figure 52B Includes figures depicting the average tumor burden. Total flux was measured on a linear scale (upper panel) and a logarithmic scale (lower panel).

[0470] Figure 53 It is a figure depicting the editing efficiency of CD5 gRNA candidates.

[0471] Figure 54 It is a graph depicting the editing efficiency obtained by NGS via CD5 gRNA candidates g103 and g104 in combination with BE4.

[0472] Figure 55 It is a schematic diagram depicting the production of an exemplary CD5 CAR construct.

[0473] Figure 56 It is a graph depicting CAR expression from lentiviral vector (LVV) screening using CD5 gRNA candidates g103 and g104.

[0474] Figure 57 It is a graph depicting CAR LVV expression based on the hinge sequence.

[0475] Figures 58A to 58D It is a graph depicting that in a live imaging cell killing assay, the edited and unedited constructs are completely cytotoxic to CD5+CCRF.

[0476] Figure 59A and Figure 59B It is a graph depicting the production of cytokine (IFNγ) by CD5 CAR LVV constructs in the presence of CD5+CCRF-CEM leukemia cell lines using sgRNA 103 or sgRNA 104. Figure 59A (Left) It is a graph depicting the production of IFNγ by T cells transduced with CD5 CAR LVV (LV63-69) edited with sgRNA103 alone or in combination with CCRF cells. Figure 59A (Right) It is a graph depicting the production of IFNγ by T cells transduced with CD5 CAR LVV (LV63-69) edited with sgRNA 104 alone or in combination with CCRF cells. Figure 59A (Bottom) It is a graph depicting the production of IFNγ by unedited T cells transduced with CD5 CARLVV (LV63-69) or CCRF cells. Figure 59B (Top) It is a graph depicting the production of IFNγ by unedited or T cells transduced with CD5 CAR LVV (LV63-69) edited with sgRNA 103 or 104 alone. Figure 59B (Bottom) It is a graph depicting the production of IFNγ by unedited or CCRF cells transduced with CD5 CAR LVV (LV63-69) edited with sgRNA 103 or 104. Detailed Description

[0477] The present invention provides genetically modified immune cells, which have enhanced anti-tumor formation activity, anti-immunosuppression, and a 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).

[0478] In one embodiment, CAR-T cells lacking or having reduced levels of functional TRAC are administered to a subject having graft-versus-host disease (GVHD) or having a propensity to develop GVHD. In one embodiment, CAR-T cells lacking or having reduced levels of beta-2 microglobulin (B2M) are administered to a subject having host-versus-graft disease (HVGD) or having a propensity to develop HVGD.

[0479] The modification of immune effector cells to express chimeric antigen receptors and knock out or knockdown specific genes is implemented using a base editor system comprising a cytidine deaminase or an adenosine deaminase as described herein, thereby reducing the negative impact that their expression may have on immune cell function.

[0480] Autologous, patient-derived chimeric antigen receptor-T cell (CAR-T) therapy has shown significant efficacy in treating some hematological cancers. While these products have led to significant clinical benefit to patients, the need to generate personalized therapies has presented significant manufacturing challenges and economic burdens. Allogeneic gene CAR-T therapy is a potential solution developed to address these challenges, having similar clinical efficacy to autologous products while treating many patients derived from a single healthy donor, thereby substantially reducing the cost of goods and batch-to-batch variability.

[0481] 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 help reduce the risk of graft-versus-host disease and host rejection of CAR-T. However, introducing multiple DSBs simultaneously results in the final cell product containing a large number of genomic rearrangements such as balanced and unbalanced translocations, and a relatively high peak of local rearrangements (including inversions and large deletions). Moreover, as the number of simultaneous gene modifications made through the introduced DSBs increases, significant genotoxicity is observed in the treated cell population. This has the potential to significantly reduce the cell expansion potential during each production process, thereby reducing the number of patients that can be treated per healthy donor.

[0482] Base editors (BEs) are a class of emerging gene editing reagents that can efficiently and user-definedly modify target genomic DNA without generating DSBs. In this article, a method for producing allogeneic CAR-T cells is proposed by using base editing technology to reduce or eliminate detectable genomic rearrangements and simultaneously improve cell expansion. As shown herein, contrary to editing strategies that use only nucleases, simultaneous modification of one or more (e.g., the loci of one, two, three, four, five, six, seven, eight, nine, ten or more genes) by base editing results in efficient gene knockout without detectable translocation events.

[0483] In some embodiments, at least one or more genes or their regulatory elements within immune cells are modified using the base editing compositions and methods provided herein. In some embodiments, the at least one or more genes or their regulatory elements 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 their regulatory elements 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 cells comprise modifications in CD5 and in at least one or more genes or their regulatory elements selected from TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. In some embodiments, the modified immune cells comprise modifications in CD7 and in at least one or more genes or their regulatory elements selected from TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. In some embodiments, the modified immune cells comprise modifications in CD33 and in at least one or more genes or their regulatory elements selected from TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. In some embodiments, the modified immune cells comprise modifications in CD3 and in at least one or more genes or their regulatory elements selected from TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. In some embodiments, the modified immune cells comprise modifications in CD123 and in at least one or more genes or their regulatory elements selected from TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. Multiplex editing of genes can be used to create CAR-T cell therapies with improved therapeutic properties. The method addresses known limitations of multiplex-edited T cell products and is a promising development towards next-generation precision cell therapies.

[0484] On the one hand, the present invention provides a universal CAR-T cell. In some embodiments, the CAR-T cells described herein are allogeneic cells. In some embodiments, the universal CAR-T cells are allogeneic T cells that can be used to express a desired CAR and can be generally applicable regardless of the immunogenic compatibility between the donor and the recipient. The allogeneic immune cells can be derived from one or more donors. In certain embodiments, the allogeneic immune cells are derived from a single human donor. For example, the allogeneic T cells can be derived from PBMCs of a single healthy human donor. In certain embodiments, the allogeneic immune cells are derived from multiple human donors. In some embodiments, as described herein, the universal CAR-T cells can be generated by introducing synchronous modifications into multiple loci using genetic modification, for example, three, four, five, six, seven, eight, nine, ten or more loci. The modifications or synchronous modifications as described herein can be gene editing generated by a base editor, such as base editing. The base editor can be a C base editor or an A base editor. As discussed herein, base editing can be used to achieve gene disruption such that the gene is not expressed. The modifications made by base editing can be used to achieve a reduction in gene expression. In some embodiments, the base editor can be used to introduce gene modifications such that the edited gene does not produce a structurally or functionally viable protein product. In some embodiments, the modifications, such as the synchronous modifications described herein, can include gene editing, such as 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 the baseline expression level. In some embodiments, the activity or functionality of the gene product can be upregulated as a result of base editing or multiple base editing events acting in concert.

[0485] In some embodiments, the generation of universal CAR-T cells may be advantageous over autologous T cells (CAR-T), which can be difficult to generate for immediate use. Compared to autologous cell preparations, allogeneic approaches are better suited for many situations associated with the uncertainties of engineering autologous T cells to express CARs and ultimately obtaining the cell products required for transplantation in medical emergencies. However, for allogeneic T cells or "off-the-shelf" T cells, it is important to carefully navigate host reactivity to CAR-T cells (HVGD) as well as the potential hostility of allogeneic T cells to host cells (GVHD). In this scenario, base editing can be successfully used to generate multiple simultaneous gene editing events such that (a) antigen expression may be reduced or downregulated to generate fratricide-resistant immune cells; (b) platform cell types may be generated that are completely devoid of or express low levels of endogenous T cell receptors, e.g., the TCRα chain (such as base editing via TRAC), or the TCRβ chain (such as base editing via TRBC1 / TRBC2); and / or (c) the expression of antigens that may be incompatible with the host tissue system may be reduced or downregulated, and vice versa.

[0486] 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 cells can be used in place of electroporation, and such methods known in the art are thus contemplated for use in any of the methods described herein.

[0487] In some regimens, an effective amount of a modified immune effector cell (e.g., a CAR-T cell) that lacks or has a reduced level of CD2 and expresses a CD2 chimeric antigen receptor of the CD2 costimulatory domain is administered to a subject having neoplasia (e.g., T or NK cell malignancy) or having the potential to develop neoplasia. In some embodiments, the CD2-modified immune cells administered to the subject are further modified with the base editing compositions and methods provided herein in one or more genes or their regulatory elements (e.g., CD52, TRAC, PD-1).

[0488] As shown herein, the combination of base editing and CAR insertion is an available strategy for generating fratricide-resistant 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. The method addresses known limitations of CAR-T therapies and is a promising development towards the next generation of precision cell therapies.

[0489] In one experiment, the 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 a single electroporation to generate cell populations 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 double-strand breaks. Thus, BE4-treated cells also did not show any measurable translocations (large-scale genomic rearrangements), while cells subjected to the same triple editing with nucleases did show detectable genomic rearrangements.

[0490] Thus, coupling nuclease-based TRAC gene knockout with simultaneous BE-mediated knockout of two additional genes generates allogeneic gene T cell populations with minimal genomic rearrangements, enabling targeted insertion of a CAR transgene at the TRAC locus. Collectively, this demonstrates that base editing alone or in combination with single nuclease knockout and CAR insertion is a useful strategy compared to nuclease-only methods to generate allogeneic T cells with minimal genomic rearrangements. This method addresses known limitations of multiply edited T cell products and is a promising development towards next-generation precision cell therapies.

[0491] Chimeric Antigen Receptors and CAR-T Cells

[0492] The present invention provides immune cells modified with a nucleobase editor as described herein that express a chimeric antigen receptor (CAR). Modifying immune cells to express a chimeric antigen receptor can enhance the immune response activity of the immune cells, wherein the chimeric antigen receptor has affinity for an epitope on an antigen, wherein the antigen is associated with altered organismal fitness. For example, the chimeric antigen receptor can have affinity for an epitope on a protein expressed in tumor cells. Since CAR-T cells can function independently of the major histocompatibility complex (MHC), activated CAR-T cells can kill tumor cells expressing the antigen. The direct action of CAR-T cells bypasses tumor cell defense mechanisms that have evolved in response to MHC presentation of antigens to immune cells.

[0493] In some embodiments, the present invention provides immune effector cells expressing a chimeric antigen receptor that targets B cells involved in an autoimmune response (e.g., B cells of a subject that express antibodies generated against the subject's own tissues).

[0494] Some embodiments include autologous immune cell immunotherapy, where the immune cells are obtained from a subject having a disease or altered fitness characterized by cancerous or other altered cells that express surface markers. The obtained immune cells are genetically modified to express a chimeric antigen receptor and are effectively redirected against a specific antigen. Thus, in some embodiments, the immune cells are obtained from a subject in need of CAR-T immunotherapy. In some embodiments, after obtaining these autologous immune cells from the subject, the cells are cultured and modified within a short period of time. In other embodiments, autologous cells are obtained and then stored for further use. This approach may be applicable to individuals who may undergo concurrent treatments in the future that will reduce immune cell counts. In allogeneic immune cell immunotherapy, the immune cells can be obtained from a donor other than the subject to be treated. In some embodiments, the immune cells are obtained from a healthy subject or donor and are genetically modified to express a chimeric antigen receptor and are effectively redirected against a specific antigen. The immune cells are administered to the subject after being modified to express the chimeric antigen receptor for the treatment of neoplasia (e.g., leukemia). In some embodiments, the immune cells to be modified to express the chimeric antigen receptor can be obtained from an existing stock of immune cell cultures.

[0495] Immune cells and / or immune effector cells can be isolated or purified from samples 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 separates 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.

[0496] In other embodiments, the present invention provides T cells having a targeted gene knockout at the TCR constant region (TRAC), which is responsible for TCRαβ surface expression. TCRαβ-deficient CAR T cells are compatible with allogeneic immunotherapy (Qasim et al., Sci. Transl. Med. 9, eaaj2013 (2017); Valton et al., Mol Ther. 2015 Sep; 23(9):1507–1518). If desired, residual TCRαβ T cells are depleted using CliniMACS 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 minor histocompatibility antigens expressed on recipient hematopoietic cells, thereby minimizing the risk of graft-versus-host disease (GVHD), which is a major 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 fluorescence-activated cell sorting, fluorescently labeled antibodies that are affinity for immune effector cell markers are used to label immune effector cells in a sample. Gating strategies suitable for cells expressing the marker are used to isolate the cells. For example, T cells can be separated from other cells in the sample by using, for example, fluorescently labeled antibodies specific for immune effector cell markers (e.g., CD4, CD8, CD28, CD45) and corresponding gating strategies. In one embodiment, a CD45 gating strategy is employed. In some embodiments, a gating strategy specific for other immune effector cells is employed instead of or in combination with the CD45 gating strategy. In one embodiment, a CD4 gating strategy is employed. In one embodiment, a CD8 gating strategy is employed. In one embodiment, a CD25 gating strategy is employed. In one embodiment, CD4 and CD8 gating strategies are employed. In one embodiment, CD4 and CD26 gating strategies are employed. In some embodiments, a gating strategy specific for other immune effector cells is employed instead of or in combination with the CD4, CD25, and / or CD8 gating strategies. In some embodiments, the Figure 40 gating strategy provided in

[0497] The immune effector cells contemplated by the present invention 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 (Th1), T helper 2 (Th2) cells, or helper T cells expressing CD4 (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 may express, in addition to the chimeric antigen receptor, exogenous cytokines, different chimeric receptors, or any other agent that will enhance the signaling or function of the immune effector cells. For example, co-expression of the chimeric antigen receptor and a cytokine can enhance the ability of CAR-T cells to lyse target cells.

[0498] The chimeric antigen receptor contemplated by the present invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. Binding of an antigen to the extracellular binding domain activates the CAR-T cells and generates an effector response, which includes CAR-T cell proliferation, cytokine production, and other processes leading to the death of antigen-expressing cells. In some embodiments of the present invention, the chimeric antigen receptor further comprises a linker. In some embodiments, the linker is a (GGGGS)n linker. In some embodiments, the linker is a (GGGGS)3 linker. In some embodiments, the CAR of the present invention includes a leader peptide sequence (e.g., the N-terminus of the antigen-binding domain). An exemplary leader peptide amino acid sequence is: METDTLLLWVLLLWVPGSTG.

[0499] The extracellular binding domain of the chimeric antigen receptor contemplated herein comprises the amino acid sequence of an antibody or an antigen-binding fragment thereof, which 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).

[0500] 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).

[0501] In various embodiments, the CAR specifically binds to Ax1. Exemplary anti-Axl CARs include, but are not limited to, CCT-301-38 (F1 Oncology Inc).

[0502] In various embodiments, the CAR specifically binds to B7H6. Exemplary anti-B7H6 CARs include, but are not limited to, CYAD-04 (Celyad SA).

[0503] In various embodiments, the CAR specifically binds to 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 (Nanj ing 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:

[0504] MALPVTALLLPLALLLHAARPDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0505] In various embodiments, the CAR specifically binds to CCK2R. Exemplary anti-CCK2R CARs include, but are not limited to, anti-CCK2R CAR-T adaptor molecule (CAM) + anti-FITC CAR T cell therapy (cancer), Endocyte / Purdue (Purdue University).

[0506] In various embodiments, the CAR specifically binds to a CD antigen. Exemplary anti-CD antigen CARs include, but are not limited to, VM-802 (ViroMed Co Ltd).

[0507] In various embodiments, the CAR specifically binds to 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).

[0508] In various embodiments, the CAR specifically binds to CD133. Exemplary anti-CD133 CARs include, but are not limited to, KD-030 (Nanjing Kaedi Biotech Inc).

[0509] In various embodiments, the CAR specifically binds to 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).

[0510] In various embodiments, the CAR specifically binds to CD171. Exemplary anti-CD171 CARs include, but are not limited to, JCAR-023 (Juno Therapeutics Inc).

[0511] In various embodiments, the CAR specifically binds to 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 Institute of Biomedicine and Health), 4G7-CARD (University College London), 4SCAR19 (Shenzhen Institute of Immunogene Therapy), 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 Bio-Gene Technology Co., Ltd), BinD-19 (Shenzhen BinDeBio Ltd.), BPX-401 (Bellicum Pharmaceuticals Inc), CAR19h28TM41BBz (Westmead Institute for Medical Research), C-CAR-011 (Chinese PLA General Hospital), 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 (iCell Gene Therapeutics), lisocabtagene maraleucel (Juno Therapeutics Inc), MatchCART (Shanghai HrainBiotechnology), 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 Lifetechnology Co Ltd), RB-1916 (Refuge Biotechnologies Inc), SKLB-083019 (Chengdu Galaxy Biopharmaceuticals Co Ltd), spCART-19 (Shanghai Youkang Biopharmaceutical 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 leraleucel (Memorial Sloan-Kettering Cancer Center), XLCART-001 (Nanjing Medical University), and yinnuokati-19 (Shenzhen Innovation Immunology Technology Co Ltd).

[0512] In various embodiments, the CAR specifically binds to CD2. Exemplary anti-CD2 CARs include, but are not limited to, UCART-2 (Wugen Inc).

[0513] In various embodiments, the CAR specifically binds to 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 (Chinese PLA General Hospital), MB-106 (Fred Hutchinson Cancer Research Center), and MB-CART20.1 (Miltenyi Biotec GmbH).

[0514] In various embodiments, the CAR specifically binds to 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 Youkang Biopharmaceutical Technology Co., Ltd.), JCAR-018 (Opus Bio Inc), MendCART (Shanghai Hrain Biotechnology), and UCART-22 (Cellectis SA).

[0515] In various embodiments, the CAR specifically binds to CD30. Exemplary anti-CD30 CARs include, but are not limited to, ATLCAR.CD30 (UNC Lineberger Comprehensive Cancer Center), CBM-C30.1 (Chinese PLA General Hospital), and Hu30-CD28zeta (National Cancer Institute).

[0516] In various embodiments, the CAR specifically binds to CD33. Exemplary anti-CD33 CARs include, but are not limited to, anti-CD33 CAR γδ T cell therapy (acute myeloid leukemia), TC BioPharm / University College London, CAR33VH (Opus Bio Inc), CART-33 (Chinese PLA General Hospital), CIK-CAR.CD33 (Formula Pharmaceuticals Inc), UCART-33 (Cellectis SA), and VOR-33 (Columbia University).

[0517] In various embodiments, the CAR specifically binds to CD38. Exemplary anti-CD38 CARs include, but are not limited to, UCART-38 (Cellectis SA).

[0518] In various embodiments, the CAR specifically binds to CD38 A2. Exemplary anti-CD38 A2 CARs include, but are not limited to, T-007 (TNK Therapeutics Inc).

[0519] In various embodiments, the CAR specifically binds to CD4. Exemplary anti-CD4 CARs include, but are not limited to, CD4CAR (iCell Gene Therapeutics).

[0520] In various embodiments, the CAR specifically binds to CD44. Exemplary anti-CD44 CARs include, but are not limited to, CAR-CD44v6 (Istituto Scientifico H San Raffaele).

[0521] In various embodiments, the CAR specifically binds to CD5. Exemplary anti-CD5 CARs include, but are not limited to, CD5CAR (iCell Gene Therapeutics). Exemplary CD5 CAR nucleic acid sequences are provided below:

[0522]

[0523]

[0524]

[0525] In various embodiments, the CAR specifically binds to CD7. Exemplary anti-CD7 CARs include, but are not limited to, CAR-pNK (PersonGen Biomedicine(Suzhou)Co Ltd) and CD7.CAR / 28ζCAR T cells (Baylor College of Medicine), UCART7 (Washington University in St Louis). The amino acid sequences of the hereditary CD7 CAR are as follows:

[0526]

[0527]

[0528] In various embodiments, the CAR specifically binds to CDH17. Exemplary anti-CDH17 CARs include, but are not limited to, ARB-001.T (Arbele Ltd).

[0529] In various embodiments, the CAR specifically binds to CEA. Exemplary anti-CEA CARs include, but are not limited to, HORC-020 (HumOrigin Inc).

[0530] In various embodiments, the CAR specifically binds to the chimeric TGF-β receptor (CTBR). Exemplary anti-chimeric TGF-β receptor (CTBR) CARs include, but are not limited to, CAR-CTBR T cells (bluebird bio Inc).

[0531] In various embodiments, the CAR specifically binds to Claudin18.2. Exemplary anti-Claudin18.2 CARs include, but are not limited to, CAR-CLD18 T cells (Carsgen Therapeutics Ltd) and KD-022 (Nanj ing KaediBiotech Inc).

[0532] In various embodiments, the CAR specifically binds to CLL1. Exemplary anti-CLL1 CARs include, but are not limited to, KITE-796 (Kite Pharma Inc).

[0533] In various embodiments, the CAR specifically binds to DLL3. Exemplary anti-DLL3 CARs include, but are not limited to, AMG-119 (Amgen Inc).

[0534] In various embodiments, the CAR specifically binds to dual BCMA / TACI (APRIL). Exemplary anti-dual BCMA / TACI (APRIL) CARs include, but are not limited to, AUTO-2 (Autolus Therapeutics Limited).

[0535] In various embodiments, the CAR specifically binds to dual CD19 / CD22. Exemplary anti-dual CD19 / CD22 CARs include, but are not limited to, AUTO-3 (Autolus Therapeutics Limited) and LCAR-L10D (Nanj ingLegend Biotech Co Ltd).

[0536] In various embodiments, the CAR specifically binds to CD19.

[0537] In various embodiments, the CAR specifically binds to dual CLL1 / CD33. Exemplary anti-dual CLL1 / CD33 CARs include, but are not limited to, ICG-136 (iCell Gene Therapeutics).

[0538] In various embodiments, the CAR specifically binds to dual EpCAM / CD3. Exemplary anti-dual EpCAM / CD3 CARs include, but are not limited to, IKT-701 (Icell Kealex Therapeutics).

[0539] In various embodiments, the CAR specifically binds to dual ErbB / 4ab. Exemplary anti-dual ErbB / 4ab CARs include, but are not limited to, LEU-001 (King's College London).

[0540] In various embodiments, the CAR specifically binds to dual FAP / CD3. Exemplary anti-dual FAP / CD3 CARs include, but are not limited to, IKT-702 (Icell Kealex Therapeutics).

[0541] In various embodiments, the CAR specifically binds to EBV. Exemplary anti-EBV CARs include, but are not limited to, TT-18 (Tessa Therapeutics Pte Ltd).

[0542] In various embodiments, the CAR specifically binds to 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).

[0543] In various embodiments, the CAR specifically binds to EGFRvIII. Exemplary anti-EGFRvIII CARs include, but are not limited to, KD-035 (Nanjing Kaedi Biotech Inc) and UCART-EgfrVIII (Cellectis SA).

[0544] In various embodiments, the CAR specifically binds to Flt3. Exemplary anti-Flt3 CARs include, but are not limited to, ALLO-819 (Pfizer Inc) and AMG-553 (Amgen Inc).

[0545] In various embodiments, the CAR specifically binds to the folate receptor. Exemplary anti-folate receptor CARs include, but are not limited to, EC17 / CAR T (Endocyte Inc).

[0546] In various embodiments, the CAR specifically binds to 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).

[0547] In various embodiments, the CAR specifically binds to 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).

[0548] In various embodiments, the CAR specifically binds to GD2 and MUC1. Exemplary anti-GD2 / MUC1 CARs include, but are not limited to, PSMACAR-T (University of Pennsylvania).

[0549] In various embodiments, the CAR specifically binds to 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).

[0550] In various embodiments, the CAR specifically binds to 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).

[0551] In various embodiments, the CAR specifically binds to IL13R. Exemplary anti-IL13R CARs include, but are not limited to, MB-101 (City of Hope) and YYB-103 (YooYoung Pharmaceuticals Co Ltd).

[0552] In various embodiments, the CAR specifically binds to integrin β-7. Exemplary anti-integrin β-7 CARs include, but are not limited to, the MMG49 CAR T cell therapy (Osaka University).

[0553] In various embodiments, the CAR specifically binds to the LC antigen. Exemplary anti-LC antigen CARs include, but are not limited to, VM-803 (ViroMed Co Ltd) and VM-804 (ViroMed Co Ltd).

[0554] In various embodiments, the CAR specifically binds to 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 KaediBiotech 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).

[0555] In various embodiments, the CAR specifically binds to 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).

[0556] In various embodiments, the CAR specifically binds to MUC16. Exemplary anti-MUC16 CARs include, but are not limited to, 4H1128Z-E27 (Eureka Therapeutics Inc) and JCAR-020 (Memorial Sloan-Kettering Cancer Center).

[0557] In various embodiments, the CAR specifically binds to nfP2X7. Exemplary anti-nfP2X7 CARs include, but are not limited to, BIL-022c (Biosceptre International Ltd).

[0558] In various embodiments, the CAR specifically binds to PSCA. Exemplary anti-PSCA CARs include, but are not limited to, BPX-601 (Bellicum Pharmaceuticals Inc).

[0559] In various embodiments, the CAR specifically binds to PSMA. CIK-CAR.PSMA (Formula Pharmaceuticals Inc) and P-PSMA-101 (Poseida Therapeutics Inc).

[0560] In various embodiments, the CAR specifically binds to ROR1. Exemplary anti-ROR1 CARs include, but are not limited to, JCAR-024 (Fred Hutchinson Cancer Research Center).

[0561] In various embodiments, the CAR specifically binds to ROR2. Exemplary anti-ROR2 CARs include, but are not limited to, CCT-301-59 (F1 Oncology Inc).

[0562] In various embodiments, the CAR specifically binds to SLAMF7. Exemplary anti-SLAMF7 CARs include, but are not limited to, UCART-CS1 (Cellectis SA).

[0563] In various embodiments, the CAR specifically binds to TRBC1. Exemplary anti-TRBC1 CARs include, but are not limited to, AUTO-4 (Autolus Therapeutics Limited).

[0564] In various embodiments, the CAR specifically binds to TRBC2. Exemplary anti-TRBC2 CARs include, but are not limited to, AUTO-5 (Autolus Therapeutics Limited).

[0565] In various embodiments, the CAR specifically binds to TSHR. Exemplary anti-TSHR CARs include, but are not limited to, ICTCAT-023 (Innovative Cellular Therapeutics Co Ltd).

[0566] 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).

[0567] 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 (Cell Design 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).

[0568] The present disclosure also provides nucleic acids encoding the chimeric antigen receptors described herein. In some embodiments, the nucleic acids are isolated or purified. Indirect in vivo delivery of the nucleic acids can be accomplished 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. The vector can then be used to transform recipient immune cells such that the 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 by reference in its entirety. In addition, the methods and vectors described herein for delivering nucleic acids encoding base editors are applicable to delivering nucleic acids encoding chimeric antigen receptors.

[0569] Extracellular binding domain

[0570] 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 the amino acid sequence of an antibody or an antigen-binding fragment thereof that 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.

[0571] In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of an antibody. In some embodiments, the chimeric antigen receptor comprises the 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 the antigen. In some embodiments, the antibody fragment portion of the chimeric antigen receptor is a single-chain variable fragment (scFv). The scFv comprises 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 that 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.

[0572] 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 joined by a disulfide bridge and two light chains, wherein each light chain is joined to one of the heavy chains by a disulfide bridge. In some embodiments, the light chain comprises a constant region and a variable region. Complementary determining regions located within the variable region of the antibody are responsible for the affinity of the antibody for a particular antigen. Thus, antibodies that recognize different antigens contain different complementary determining regions. The complementary determining regions are located within the variable domain 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 and variable light chains are joined by a (GGGGS)n linker, where n is an integer from 1 to 10. In some embodiments, the linker is a (GGGGS)3 linker.

[0573] In some embodiments, the antigen recognized and bound by the extracellular domain is a protein or peptide, nucleic acid, lipid, or 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 this antigen can result in an extreme immune response. In some embodiments, the antigen is autologous and is expressed on diseased or otherwise altered cells. For example, in some embodiments, the antigen is expressed in tumor cells. In some embodiments, the tumor cells are solid tumor cells. In other embodiments, the tumor cells are liquid tumor cells. In other embodiments, the tumor cells are hematological cancers, such as B cell cancers. In some embodiments, the B cell cancer is lymphoma or leukemia.

[0574] Liquid cancers to be treated using the methods described herein can be, for example, leukemia. In some cases, leukemia includes preleukemia. In some cases, leukemia is acute leukemia. Acute leukemia includes, for example, acute myeloid leukemia (AML). Acute leukemia also includes, for example, acute lymphoblastic leukemia or acute lymphocytic leukemia (ALL); ALL includes B-lineage ALL, T-lineage ALL, and T cell acute lymphoblastic leukemia (T-ALL).

[0575] Non-limiting examples of tumor formation include 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 leukemia, angioimmunoblastic T / NK cell lymphoma, hepatosplenic T cell lymphoma, primary cutaneous CD30+ lymphoproliferative disease, extranodal NK / T cell lymphoma, adult T cell leukemia / lymphoma, T cell prolymphocytic leukemia, subcutaneous panniculitis-like T cell lymphoma, primary cutaneous γδ T cell lymphoma, aggressive NK cell leukemia, and enteropathy-associated T cell lymphoma. In some embodiments, the tumor formation is T cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the tumor formation is T cell acute myeloid leukemia (AML).

[0576] Antibody-antigen interactions are non-covalent interactions resulting from hydrogen 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 formula:

[0577] KA = [antibody-antigen] / [antibody][antigen], where

[0578] [Ab] = the molar concentration of unoccupied binding sites on the antibody;

[0579] [Ag] = the molar concentration of unoccupied binding sites on the antigen; and

[0580] [Ab-Ag] = the molar concentration of the antibody-antigen complex.

[0581] Antibody-antigen interactions can also be characterized based on the dissociation of the antigen from the antibody. The dissociation constant (KD) is the ratio of the association rate to the dissociation rate and is inversely proportional to the affinity constant. Thus, KD = 1 / KA. Those skilled in the art will be familiar with these concepts and will know that conventional methods such as ELISA assays can be used to calculate these constants.

[0582] Transmembrane domain

[0583] The chimeric antigen receptor of the present invention includes a transmembrane domain. The transmembrane domain of the chimeric antigen receptor described herein spans the lipid bilayer cell membrane of the CAR-T cell and separates the extracellular binding domain from the intracellular signaling domain. In some embodiments, this domain is derived from other receptors having a transmembrane domain, 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. "Humanized" means having a sequence of nucleic acid encoding the transmembrane domain that is optimized to be more readily or efficiently expressed in a human subject. In some embodiments, the transmembrane domain is derived from another transmembrane domain expressed in human immune effector cells. Examples of such proteins include, but are not limited to, 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 having a transmembrane domain. In some embodiments, the transmembrane domain will be synthetic and such sequences will contain a number of hydrophobic residues.

[0584] The transmembrane domain for the disclosed CAR can include at least the transmembrane regions of the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, 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, CD8α, CD28, or CD3ζ. In some embodiments, the transmembrane domain is the CD28 transmembrane domain. In some embodiments, the transmembrane domain is the CD8α transmembrane domain.

[0585] In some embodiments, the transmembrane domain is the CD8α hinge and transmembrane domain. In some embodiments, the CD8α 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:

[0586] SDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0587] In some embodiments, the chimeric antigen receptor is designed to include a spacer sequence located between the transmembrane domain and the extracellular domain, the intracellular domain, or both. Such spacer sequences 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 still 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 is used to position such linked domains to enhance or optimize chimeric antigen receptor function. In some embodiments, the hinge / spacer sequence is selected from CH3, CD8α, or CD28.

[0588] Intracellular signaling domain

[0589] The chimeric antigen receptor of the present invention includes an intracellular signaling domain. The intracellular signaling domain is the intracellular portion of a protein that is expressed within a T cell, which transduces signals for T cell effector functions (e.g., activation signals) and directs the T cell to perform specialized functions. T cell activation can be induced by a variety of factors, including the binding of an antigen to the T cell receptor on the surface of the T cell and the binding of a cognate ligand to a co-stimulatory molecule on the surface of the T cell. T cell co-stimulatory molecules are cognate binding partners on the T cell that specifically bind to co-stimulatory ligands, thereby mediating co-stimulatory responses of the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to, MHC class I molecules. Activation of the T cell results in 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 the CD3ζ, CD8, CD28, CD27, CD154, GITR (TNFRSF18), CD134 (OX40), and CD137 (4-1BB) signaling domains.

[0590] The intracellular signaling domain of the chimeric antigen receptor contemplated herein includes a primary signaling domain. In some embodiments, the chimeric antigen receptor includes a primary signaling domain and a co-stimulatory signaling domain.

[0591] 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. The ITAMs incorporated into the chimeric antigen receptor can be derived from ITAMs from other cell receptors. In some embodiments, the primary signaling domain comprising an ITAM can be derived from subunits of the TCR complex, such as CD3γ, CD3ε, CD3ζ, or CD3δ (see Figure 1A ). In some embodiments, the primary signaling domain comprising an ITAM can be derived from FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, or CD66d.

[0592] In some embodiments, the primary signaling domain is selected from the group consisting of CD8, CD28, CD134 (OX40), CD137 (4-1BB), and CD3ζ. In some embodiments, the primary signaling domain is the CD3ζ signaling domain. In some embodiments, the CD3ζ 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:

[0593] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0594] In some embodiments, the primary signaling domain is the 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:

[0595] rrdqrlppdahkppgggsfrtpiqeeqadahstlaki

[0596] In some embodiments, the secondary or co-stimulatory signaling domain is derived from 4-1BB, CD2, CD4, CD28, CDS, CD8α, CD83, CD134, CD137, ICOS, or CD154. In some embodiments, the secondary signaling domain is the 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:

[0597] SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0598] In some embodiments, the secondary signaling domain is the 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:

[0599] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0600] 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:

[0601] RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0602] In some embodiments, the CAR comprises one or more signaling domains. In some embodiments, the CAR comprises a 4-1BB signaling domain and a CD3ζ signaling domain. In some embodiments, the CAR comprises a CD28 signaling domain and a CD3ζ signaling domain.

[0603] Editing of target genes in immune cells

[0604] The present invention provides immune cells that comprise a chimeric antigen receptor (CAR) and one or more edited genes, one or more of their regulatory elements, or a combination thereof, wherein the 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 suppression or inhibition, 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 may comprise a single base edit. In some embodiments, each edited gene may comprise multiple base edits located in different regions of the gene.

[0605] 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-fold or more-fold editing can be introduced into one or more genes simultaneously. In some embodiments, immune cells, including but not limited to any immune cells comprising an edited gene selected from any of the foregoing gene edits, can be edited to generate a mutation in other genes, which enhances the function of CAR-T or reduces immunosuppression or inhibition of the cells.

[0606] 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-tumorigenic activity compared to similar CAR-T cells that do not further have one or more edits described herein.

[0607] In some embodiments, provided herein is an immune cell having at least one modification in an endogenous gene or its regulatory element. 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 their regulatory elements.

[0608] In some embodiments, the one or more genes, or one or more regulatory elements thereof, or combinations thereof are optionally 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 at least one additional edited gene. In some embodiments, the immune cell comprises an edited CD5 gene and at least one additional edited gene. In some embodiments, the immune cell comprises an edited CD7 gene and at least one additional edited gene. In some embodiments, the immune cell comprises an edited CD33 gene and at least one additional edited gene. In some embodiments, the immune cell comprises an edited CD123 gene and at least one additional 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.

[0609] In various embodiments, the modified immune cells comprise 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 cells comprise 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 cells comprise a mutation in CD5 and a mutation in one or more or a combination of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In one embodiment, the modified immune cells comprise a mutation in CD5, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0610] In various embodiments, the modified immune cells comprise 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 cells comprise 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 cells comprise a mutation in CD7 and a mutation in one or more or a combination of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In one embodiment, the modified immune cells comprise a mutation in CD7, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0611] In various embodiments, the modified immune cells comprise 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 cells comprise 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 cells comprise a mutation in CD3 and a mutation in one or more or a combination of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In one embodiment, the modified immune cells comprise a mutation in CD3, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0612] In various embodiments, the modified immune cells comprise 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 cells comprise 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 cells comprise a mutation in CD33 and a mutation in one or more or a combination of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In one embodiment, the modified immune cells comprise a mutation in CD33, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0613] In various embodiments, the modified immune cells comprise a mutation in one or more or a combination of CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cells comprise 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 cells comprise a mutation in CD123 and a mutation in one or more or a combination of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In one embodiment, the modified immune cells comprise a mutation in CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0614] In some embodiments, at least one modification is a single nucleobase modification. In some embodiments, an altered endogenous gene can be created by base editing. In some embodiments, base editing can reduce or attenuate gene expression. In some embodiments, base editing can reduce or attenuate gene activation. In some embodiments, base editing can reduce or attenuate the functionality of a gene product. In some other embodiments, base editing can activate or enhance gene expression. In some embodiments, base editing can increase the functionality of a gene product.

[0615] Allogeneic immune cells that express an endogenous immune cell receptor as well as a chimeric antigen receptor can recognize and adhere to host cells, a condition known as graft-versus-host disease (GVHD). The α component of the immune cell receptor complex is encoded by the TRAC gene, and in some embodiments, this gene is edited such that the α subunit of the TCR complex is non-functional or absent. Because this subunit is required for endogenous immune cell signaling, editing this gene can reduce the risk of graft-versus-host disease caused by allogeneic immune cells.

[0616] In some embodiments, gene editing used to provide fratricide resistance, enhance immune cell function, or reduce immune repression or inhibition can occur in the immune cells before the cells are transduced to express a chimeric antigen receptor. On the other hand, gene editing used to enhance immune cell function or reduce immune repression or inhibition can occur in CAR-T cells, i.e., after the immune cells have been transduced to express a chimeric antigen receptor.

[0617] In some embodiments of the present invention, CD5 in CAR-T cells is edited to knockout or knockdown expression. Then, the CAR-T is transformed to express a chimeric antigen receptor having a CD5 scFv. By knocking out or knocking down the expression of the CD5 gene, the modified CAR-T cells are less likely to kill each other.

[0618] In some embodiments of the present invention, CD7 in CAR-T cells is edited to knockout or knockdown expression. Then, the CAR-T is transformed to express a chimeric antigen receptor having a CD7 scFv. By knocking out or knocking down the expression of the CD7 gene, the modified CAR-T cells are less likely to kill each other.

[0619] In some embodiments of the present invention, CD33 in CAR-T cells is edited to knockout or knockdown expression. Then, the CAR-T is transformed to express a chimeric antigen receptor having a CD33 scFv. By knocking out or knocking down the expression of the CD33 gene, the modified CAR-T cells are less likely to kill each other.

[0620] In some embodiments of the present invention, CD3 in CAR-T cells is edited to knockout or knockdown expression. Then, the CAR-T is transformed to express a chimeric antigen receptor having a CD3 scFv. By knocking out or knocking down the expression of the CD3 gene, the modified CAR-T cells are less likely to kill each other.

[0621] In some embodiments of the present invention, CD123 in CAR-T cells is edited to knockout or knockdown expression. Then, the CAR-T is transformed to express a chimeric antigen receptor having a CD123 scFv. By knocking out or knocking down the expression of the CD123 gene, the modified CAR-T cells are less likely to kill each other.

[0622] Host immune cells can potentially recognize allogeneic CAR-T cells as non-self and initiate an immune response to eliminate the non-self cells. B2M is expressed in almost all nucleated cells and associates with the MHC class I complex ( Figure 1B ). Circulating host CD8+ T cells can recognize this B2M protein as non-self and kill allogeneic cells. To overcome this graft rejection, in some embodiments, the B2M gene is edited to knockout or knockdown 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.

[0623] In some embodiments, the immune cells comprise a chimeric antigen receptor and one or more edited genes, their regulatory elements, or a combination thereof. The edited gene can be an immune response regulatory gene, an immunogen gene, a checkpoint inhibitor gene, a gene involved in an immune response, a cell surface marker such as a T cell surface marker, or any combination thereof. In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited gene that is related to activated T cell proliferation, α-β T cell activation, γ-δ T cell activation, positive regulation of T cell proliferation, or negative regulation of T helper cell proliferation or differentiation, or its regulatory element or a combination thereof. In some embodiments, the edited gene can be a checkpoint inhibitor gene, such as the PD1 gene, the PDC1 gene, a member related to or regulating its formation or activation pathway. In some embodiments, the edited gene is the TRAC gene. In some embodiments, the edited gene is the CD5 gene. In some embodiments, the edited gene is the CD7 gene. In some embodiments, the edited gene is the CD33 gene. In some embodiments, the edited gene is the CD3 gene. In some embodiments, the edited gene is the CD123 gene. In some embodiments, the edited gene is the B2M gene. In some embodiments, the edited gene is the CIITA gene. In some embodiments, the edited gene is the TRBC1 / 2 gene. In some embodiments, the edited gene is the CD5 gene. In some embodiments, the edited gene is the CD7 gene. In some embodiments, the edited gene is the CD52 gene. In some embodiments, at least one edited gene is selected from 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.

[0624] In some embodiments, editing of the endogenous gene reduces the expression of the gene. In some embodiments, editing of the endogenous gene reduces the expression of the gene by at least 50% compared to control cells that have not been modified. In some embodiments, editing of the endogenous gene reduces the expression of the gene by at least 60% compared to control cells that have not been modified. In some embodiments, editing of the endogenous gene reduces the expression of the gene by at least 70% compared to control cells that have not been modified. In some embodiments, editing of the endogenous gene reduces the expression of the gene by at least 80% compared to control cells that have not been modified. In some embodiments, editing of the endogenous gene reduces the expression of the gene by at least 90% compared to control cells that have not been modified. In some embodiments, editing of the endogenous gene reduces the expression of the gene by at least 100% compared to control cells that have not been modified. In some embodiments, editing of the endogenous gene eliminates gene expression.

[0625] In some embodiments of the present invention, PDCD1 in CAR-T cells is edited to knockout or knockdown expression. The PDCD1 gene encodes the cell surface receptor PD-1 (an immune system checkpoint expressed in immune cells) and is involved in reducing immunity by promoting apoptosis of antigen-specific immune cells. By knocking out or knocking down the expression of the PDCD1 gene, the modified CAR-T cells are less likely to undergo apoptosis, more likely to proliferate, and can escape from the programmed cell death immune checkpoint.

[0626] CBLB encodes an E3 ubiquitin ligase that plays a significant role in inhibiting the activation of immune effector cells. Referring to Figure 1C , the CBLB protein favors the signaling pathway leading to immune effector cell tolerance and actively inhibits the signaling pathway leading to immune effector cell activation. Since immune effector cell activation is necessary for in vivo proliferation of CAR-T cells after transplantation, in some embodiments of the present invention, the CBLB gene is edited to knockout or knockdown expression.

[0627] In some embodiments, the present invention provides an immune cell having an edited TRAC gene (where the TRAC gene may 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, the present invention provides 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 the endogenous T cell receptor α protein.

[0628] 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 transactivator. 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 transactivator.

[0629] 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.

[0630] In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited TRAC, B2M, PDCD1, CBLB gene, or a combination thereof, wherein the expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited TRAC gene, wherein the expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC and B2M genes, wherein the expression of the edited genes is either knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC and PDCD1 genes, wherein the expression of the edited genes is either knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC and CBLB genes, wherein the expression of the edited genes is either knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, B2M, and PDCD1 genes, wherein the expression of the edited genes is either knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, B2M, and CBLB genes, wherein the expression of the edited genes is either knocked out or knocked down. In some embodiments, the immune cells or immune effector cells comprise a chimeric antigen receptor and edited TRAC, PDCD1, and CBLB genes, wherein the expression of the edited genes is either knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, B2M, PDCD1, and CBLB genes, wherein the expression of the edited genes is either knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited B2M gene, wherein the expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M and PDCD1 genes, wherein the expression of the edited genes is either knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M and CBLB genes, wherein the expression of the edited genes is either knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M, PDCD1, and CBLB genes, wherein the expression of the edited genes is either knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited PDCD gene, wherein the expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited PDCD and CBLB genes, wherein the expression of the edited genes is either knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited CBLB gene, wherein the expression of the edited gene is knocked out or knocked down.

[0631] In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited CD5 gene, wherein the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the expression of the edited gene is knocked out or knocked down.

[0632] Base editing can be located at any suitable position of a gene, or in the regulatory elements of the gene. Thus, it is expected that a single-base edit at the start codon, for example, can completely abolish the expression of the gene. In some embodiments, the altered endogenous gene can be modified or edited in an exon, an intron, an exon-intron junction, or its regulatory elements. The modification can be an edit of a single nucleobase in the gene or its regulatory elements. 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 in an untruncated region of the gene, for example, in the 3'-UTR or 5'-UTR. In some embodiments, the modification is in the regulatory elements of the endogenous gene. In some embodiments, the modification is in a promoter, an enhancer, an operator, a silencer, an insulator, a terminator, a transcription start sequence, a translation start sequence (e.g., Kozak sequence), or a combination thereof. In some embodiments, base editing can introduce a premature STOP codon into an exon, resulting in the absence or truncation of a translation product that may misfold and thus be eliminated by degradation, or base editing can produce an unstable mRNA that is readily degraded.

[0633] In some embodiments, base editing can be performed, for example, on exon 1, exon 2, exon 3, 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 actions 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.

[0634] In some embodiments, base editing in the human PDC1 / PD-1A gene is performed by editing positions 4, 6, 7, 8, or 9 of the guide RNA spacer targeting exon 1. In some embodiments, base editing in the human PDC1 / PD-1A gene is performed by editing positions 4, 6, 7, 8, or 9 of the guide RNA spacer targeting exon 1. In some embodiments, base editing in the human PDC1 / PD-1A gene is performed by editing positions 7, 8, or 9 of the guide RNA spacer targeting exon 2. In some embodiments, base editing in the human PDC1 / PD-1A gene is performed by editing positions 5, 7, or 8 of the guide RNA spacer targeting exon 3. In some embodiments, base editing in the human PDC1 / PD-1A gene is performed by editing positions 5 or 8 of the guide RNA spacer targeting exon 5.

[0635] 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 actions 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 positions 4, 8, 9 within exon 1. In some embodiments, base editing in the human CD7 gene is performed by editing positions 5, 6, 7, 8, or 9 of the guide RNA spacer targeting exon 2. In some embodiments, base editing in the human CD7 gene is performed by editing positions 4 or 9 of the guide RNA spacer targeting exon 3.

[0636] 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 actions 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 the guide RNA spacer targeting exon 1. In some embodiments, base editing in the human LAG-3 gene is performed by editing positions 4, 6, or 8 of the guide RNA spacer targeting exon 2. In some embodiments, base editing in the human LAG-3 gene is performed by editing positions 4, 5, 6, or 7 of the guide RNA spacer targeting exon 3. In some embodiments, base editing in the human LAG-3 gene is performed by editing positions 4, 8, or 9 of the guide RNA spacer targeting exon 4. In some embodiments, base editing in the human LAG-3 gene is performed by editing positions 8 or 9 of the guide RNA spacer targeting exon 5. In some embodiments, base editing in the human LAG-3 gene is performed by editing positions 4, 6, 7, or 8 of the guide RNA spacer targeting exon 6. In some embodiments, base editing in the human LAG-3 gene is performed by editing positions 4, 6, or 7 of the guide RNA spacer targeting exon 7. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 8 of the guide RNA spacer targeting exon 8.

[0637] 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 the guide RNA spacer targeting exon 9. In some embodiments, base editing in the human CD33 gene is performed by editing position 4, 5, 6, or 8 of the guide RNA spacer targeting exon 2. In some embodiments, base editing in the human CD33 gene is performed by editing position 4, 5, 6, or 7 of the guide RNA spacer targeting exon 3. In some embodiments, base editing in the human CD33 gene is performed by editing position 6 or 7 of the guide RNA spacer targeting exon 4. In some embodiments, base editing in the human CD33 gene is performed by editing position 7 or 8 of the guide RNA spacer targeting exon 5. In some embodiments, base editing in the human CD33 gene is performed by editing position 4, 5, or 6 of the guide RNA spacer targeting exon 6.

[0638] 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 actions 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 combination thereof. In some embodiments, base editing in the human CD123 gene is performed by editing position 1 of the guide RNA spacer targeting exon 6. In some embodiments, base editing in the human CD123 gene is performed by editing positions 4, 6, or 8 of the guide RNA spacer targeting exon 2. In some embodiments, base editing in the human CD123 gene is performed by editing positions 5, 6, or 8 of the guide RNA spacer targeting exon 3. In some embodiments, base editing in the human CD123 gene is performed by editing positions 5 or 6 of the guide RNA spacer targeting exon 4. In some embodiments, base editing in the human CD123 gene is performed by editing positions 4 or 5 of the guide RNA spacer targeting exon 5. In some embodiments, base editing in the human CD123 gene is performed by editing position 5 of the guide RNA spacer targeting exon 7. In some embodiments, base editing in the human CD123 gene is performed by editing positions 5, 6, 7, or 8 of the guide RNA spacer targeting exon 8. In some embodiments, base editing in the human CD123 gene is performed by editing positions 4, 7, or 8 of the guide RNA spacer targeting exon 10. In some embodiments, base editing in the human CD123 gene is performed by editing positions 5 or 8 of the guide RNA spacer targeting exon 11.

[0639] In some embodiments, base editing can be performed, for example, on exon 1, or exon 3, or exon 4, or exon 5, or exon 6, or exon 7, or exon 8, or exon 9 of the human FAS gene. In some embodiments, base editing in the human FAS gene is performed at a site within exon 1. In some embodiments, base editing in the human FAS gene is performed at a site within exon 3. In some embodiments, base editing in the human FAS gene is performed at a site within exon 4. In some embodiments, base editing in the human FAS gene is performed at a site within exon 5. In some embodiments, base editing in the human FAS gene is performed at a site within exon 6. In some embodiments, base editing in the human FAS gene is performed at a site within exon 7. In some embodiments, base editing in the human FAS gene is performed at a site within exon 8. In some embodiments, base editing in the human FAS gene is performed at a site within exon 9. In some embodiments, one or more base editing operations can be performed on the human FAS gene at exon 1, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, or any combination thereof.

[0640] In some embodiments, base editing in the human FAS gene is performed by editing position 9 of the guide RNA spacer targeting exon 1. In some embodiments, base editing in the human FAS gene is performed by editing position 6 of the guide RNA spacer targeting exon 3. In some embodiments, base editing in the human FAS gen...

Claims

1. An ex vivo method for producing CAR-expressing immune cells or populations of CAR-expressing immune cells with reduced immunogenicity, the method comprising: a) contacting a target polynucleotide in the immune cells or population of immune cells with 1) a base editor system comprising a guide polynucleotide and a base editor, the base editor comprising a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a TadA*8 deaminase domain; or 2) a base editor system comprising a guide polynucleotide, napDNAbp and a TadA*8 deaminase domain, wherein the amino acid sequence of the TadA*8 deaminase domain is as shown in SEQ ID NO:8 or as shown at positions 2 to 167 of SEQ ID NO:8, and the amino acid sequence of the TadA*8 deaminase domain has amino acid alterations with reference to SEQ ID NO:8, wherein the amino acid alterations consist of: Y147T; Y147R; Q154S; Y123H; V82S; T166R; Q154R; Y147R, Q154R and Y123H; Y147R, Q154R and I76Y; Y147R, Q154R and T166R; Y147T and Q154R; Y147T and Q154S; I76Y, Y123H, Y147R and Q154R; I76Y and V82S; V82S and Y147R; V82S, Y123H and Y147R; V82S and Q154R; V82S, Y123H and Q154R; V82S, Y123H, Y147R and Q154R; I76Y, V82S, Y123H, Y147R and Q154R; Y147R and Q154S; V82S and Q154S; V82S and Y123H; or V82S, Y123H and Y147T; b) introducing, by nucleobase modification, mutations that disrupt splice sites in the CD5 gene and / or the CD7 gene, thereby reducing or eliminating the expression of antigens 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, by multiplex editing, mutations that disrupt splice sites in the B2M gene, the CD52 gene, the PDl gene and / or the TRAC gene into the immune cells or population of immune cells, thereby reducing or eliminating the expression of the following groups of polypeptides encoded by the genes, wherein the B2M gene encodes the amino acid sequence of SEQ ID NO:17, the CD52 gene encodes the amino acid sequence of SEQ ID NO:33, the Dl gene encodes the amino acid sequence of SEQ ID NO:91, and the TRAC gene encodes the amino acid sequence of SEQID NO:93; wherein the groups of polypeptides are: i) B2M, TRAC and PD1, or ii) PD1, TRAC, and CD52; and d) expressing a chimeric antigen receptor targeting a CD5 or CD7 antigen with reduced or eliminated expression in the immune cell or population of immune cells, thereby producing a CAR-expressing immune cell or population of CAR-expressing immune cells with reduced immunogenicity, wherein the CAR-expressing immune cell or population of CAR-expressing immune cells produced by the method does not contain a detectable translocation, and wherein 1) the method comprises reducing or eliminating the expression of the CD7 antigen, and wherein the CAR targets the CD7 antigen and consists of the amino acid sequence shown in SEQ ID NO: 113; or 2) the CAR is a CD5 chimeric antigen receptor consisting of an amino acid sequence selected from SEQ ID NOs: 3 to 7 and 112.

2. The method according to claim 1, wherein the guide polynucleotide comprises a nucleic acid sequence selected from CUCUUACCUGUACCAUAACC, CCUACCUGUCACCAGGACCA, and CACCUACCUAAGAACCAUCC.

3. The method according to claim 1, wherein the CAR-expressing immune cell or population of CAR-expressing immune cells produced by the method exhibits fratricide resistance and / or increased antitumor formation activity compared to corresponding control cells.

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

5. The method according to 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.

6. The method according to claim 1, further comprising depleting TCRα / β+ cells from the CAR-expressing immune cell or population of CAR-expressing immune cells.

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