CBLB endonuclease variants, compositions, and methods of use

Abstract

To provide improved genome editing compositions and methods for editing the CBLB gene.SOLUTION: The disclosure further provides a genome edited cell for the prevention, treatment, or amelioration of at least one symptom of cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency. In various embodiments, the present disclosure contemplates, in part, a polypeptide comprising a homing endonuclease (HE) variant that cleaves a target site in the human CBLB gene.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 567,417, filed October 3, 2017, and U.S. Provisional Patent Application No. 62 / 511,194, filed May 25, 2017, each of which is incorporated by reference herein in its entirety.

[0002] Explanation of Sequence Listing The sequence listing accompanying this application is provided in text format in lieu of a hard copy and is hereby incorporated by reference. The text file containing the sequence listing is named BLBD_087_02WO_ST25.txt. This text file is 152 KB, was created on May 25, 2018, and is being submitted electronically via EFS-Web simultaneously with the filing of this application.

[0003] The present disclosure relates to improved genome editing compositions. More specifically, the present disclosure relates to nuclease variants, compositions, and methods of use thereof for editing the human Casitas B-lineage (Cbl) lymphoma proto-oncogene B (CBLB) gene. [Background technology]

[0004] The global burden of cancer doubled between 1975 and 2000. Cancer is the second leading cause of morbidity and mortality worldwide, with approximately 14.1 million new cases and 8.2 million cancer-related deaths in 2012. The most common cancers are breast cancer, lung and bronchial cancer, prostate cancer, colon and rectal cancer, bladder cancer, melanoma of the skin, non-Hodgkin's lymphoma, thyroid cancer, kidney and renal pelvis cancer, endometrial cancer, leukemia, and pancreatic cancer. The number of new cancer cases is expected to rise to 22 million within the next 20 years.

[0005] The immune system plays a major role in the detection and eradication of human cancer. The majority of transformed cells are rapidly detected by immune sentinels and destroyed through the activation of antigen-specific T cells via clonally expressed T cell receptors (TCRs). Therefore, cancer can be viewed as an immunological disorder—a malfunction of the immune system to mount the antitumor responses necessary to permanently suppress and eliminate disease. To more effectively eradicate cancer, certain immunotherapeutic interventions developed over the past few decades have focused specifically on enhancing T cell immunity. These treatments have only resulted in isolated cases of disease remission, without achieving substantial overall responses.

[0006] More recently, adoptive cell therapy strategies based on the isolation, modification, expansion, and reinfusion of T cells have been explored and tested in early-phase clinical trials. T cells have often become the effector cells of choice for cancer immunotherapy due to their selective recognition and potent effector mechanisms. These treatments have shown mixed response rates, although a small number of patients have experienced durable remissions, highlighting the as-yet-understood potential of T cell-based immunotherapy.

[0007] Successful recognition of tumor cell-associated antigens by cytolytic T cells initiates the lysis of targeted tumors and underpins any effective cancer immunotherapy approach. Tumor-infiltrating T cells (TILs) express TCRs specifically directed against tumor-associated antigens, but substantial numbers of TILs are limited to only a few human cancers. Genetically engineered T cell receptors (TCRs) and chimeric antigen receptors (CARs) have the potential to increase the applicability of T cell-based immunotherapy to many cancers and other immune disorders.

[0008] Furthermore, state-of-the-art genetically engineered T cells are still regulated by a complex immunosuppressive tumor microenvironment consisting of cancer cells, inflammatory cells, stromal cells, and cytokines. Among these components, cancer cells, inflammatory cells, and suppressive cytokines adversely affect T cell phenotype and function. Collectively, the tumor microenvironment drives T cells to ultimately differentiate into exhausted T cells.

[0009] T cell exhaustion is a state of T cell dysfunction in a chronic environment characterized by increased expression of or increased signaling by inhibitory receptors, reduced effector cytokine production, and a diminished ability to sustainably eliminate cancer. Exhausted T cells also exhibit a hierarchical loss of function: reduced IL-2 production and ex vivo killing ability are lost in the early stages of exhaustion, TNF-α production is lost in the intermediate stages, and IFN-γ and GzmB production are lost in the later stages of exhaustion. Most T cells in the tumor microenvironment differentiate into exhausted T cells, lose their ability to eliminate cancer, and are eventually eliminated.

[0010] To date, there are no demonstrable clinical examples of adoptive cell therapy with increased persistence and tolerance to the immunosuppressive tumor microenvironment. Summary of the Invention [Means for solving the problem]

[0011] The present disclosure generally relates, in part, to compositions comprising a homing endonuclease variant that cleaves a target site in the human casitas lineage B (Cbl) lymphoma proto-oncogene B (CBLB) gene and a megaTAL.

[0012] In various embodiments, the present disclosure contemplates, in part, a polypeptide comprising a homing endonuclease (HE) variant that cleaves a target site within the human CBLB gene.

[0013] In certain embodiments, the HE variant is a LAGLIDADG homing endonuclease (LHE) variant.

[0014] In some embodiments, the polypeptide comprises a biologically active fragment of an HE variant.

[0015] In certain embodiments, a biologically active fragment lacks 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal amino acids compared to the corresponding wild-type HE.

[0016] In some embodiments, the biologically active fragment lacks four N-terminal amino acids compared to the corresponding wild-type HE.

[0017] In a further embodiment, the biologically active fragment lacks 8 N-terminal amino acids compared to the corresponding wild-type HE.

[0018] In certain embodiments, a biologically active fragment lacks 1, 2, 3, 4, 5, or 6 C-terminal amino acids compared to the corresponding wild-type HE.

[0019] In certain embodiments, the biologically active fragment lacks C-terminal amino acids compared to the corresponding wild-type HE.

[0020] In additional embodiments, the biologically active fragment lacks two C-terminal amino acids compared to the corresponding wild-type HE.

[0021] In certain embodiments, the HE variant is a variant of LHE selected from the group consisting of I-CreI and I-SceI.

[0022] In certain embodiments, the HE variants are I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII , I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, and I-Vdi141I.

[0023] In some embodiments, the HE variant is a variant of LHE selected from the group consisting of I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, and SmaMI.

[0024] In additional embodiments, the HE variant is an I-OnuI LHE variant.

[0025] In certain embodiments, the HE variant comprises one or more amino acid substitutions in the DNA recognition interface at amino acid positions selected from the group consisting of 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76, 78, 80, 82, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of the I-OnuI LHE amino acid sequence set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.

[0026] In certain embodiments, the HE variant comprises at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions in the DNA recognition interface at amino acid positions selected from the group consisting of 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76, 78, 80, 82, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of the I-OnuI LHE amino acid sequence set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.

[0027] In certain embodiments, the HE variant comprises one or more amino acid substitutions at amino acid positions selected from the group consisting of 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76, 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of the I-OnuI LHE amino acid sequence set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.

[0028] In certain embodiments, the HE variants have at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions in the I-OnuI sequence shown in SEQ ID NOs: 1-5. Comprising an LHE amino acid sequence or a biologically active fragment thereof at an amino acid position selected from the group consisting of 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76, 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240.

[0029] In certain embodiments, the HE variant comprises at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions at at least one position selected from the group consisting of positions 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 78, 80, 92, 116, 138, 143, 159, 168, 178, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 207, 223, 225, 227, 232, 236, and 238 of any one of SEQ ID NOs: 1-5, or a biologically active fragment thereof.

[0030] In some embodiments, the HE variant has one of the following amino acid substitutions in any one of SEQ ID NOS: 1-5, or a biologically active fragment thereof: S24C, L26R, L26G, R28D, R28Y, R30H, N32A, N32S, K34D, K34V, S35L, S36R, V37A, V37S, S40R, E42R, G44A, G44S, Q46E, T48V, T48S, V68T, V68K, A70Y, S72A, S78R, K80Q, D92G, V116L, L138M, T143N, S159P, F168L, E178D, C180 and at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of S, F182V, F182M, N184E, I186K, I186M, S188R, S188N, K189R, S190N, K191P, K191N, L192V, G193K, G193I, Q195G, Q195R, Q197R, V199R, S201G, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0031] In further embodiments, the HE variant has the following amino acid substitutions in any one of SEQ ID NOs: 1-5 or a biologically active fragment thereof: S24C, L26R, R28D, N32A, K34D, S35L, S36R, V37A, S40R, E42R, G44A, Q46E, T48V, V68T, A70Y, S72A, S78R, K80Q, L138M, T143N, F168L, E178D, C180S, F18 At least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of 2V, N184E, I186K, S188R, K189R, K191P, L192V, G193K, Q195G, Q197R, V199R, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0032] In certain embodiments, the HE variant has one of the following amino acid substitutions in any one of SEQ ID NOS: 1-5, or a biologically active fragment thereof: S24C, L26R, R28D, N32A, K34D, S35L, S36R, V37A, S40R, E42R, G44A, Q46E, T48V, V68T, A70Y, S72A, S78R, K80Q, L138M, T143N, S159P, F168L, E178D, C180S, F18 At least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of 2M, N184E, I186M, S188N, S190N, K191N, L192V, G193I, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0033] In certain embodiments, the HE variant has one of the following amino acid substitutions in any one of SEQ ID NOS: 1-5, or a biologically active fragment thereof: S24C, L26R, R28D, N32A, K34D, S35L, S36R, V37A, S40R, E42R, G44S, Q46E, T48S, V68T, A70Y, S72A, S78R, K80Q, D92G, V116L, L138M, T143N, S159P, F168L, E178D, C18 The nucleic acid sequence of the present invention includes at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the amino acid sequences selected from the group consisting of nucleotides 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 110, 111, 112, 113

[0034] In some embodiments, the HE variant has one of the following amino acid substitutions in any one of SEQ ID NOS: 1-5, or a biologically active fragment thereof: S24C, L26R, R28D, R30H, N32A, K34V, S35L, S36R, V37S, S40R, E42R, G44S, Q46E, T48V, V68T, V68K, A70Y, S72A, S78R, K80Q, L138M, T143N, S159P, F168L, E178D, C18 The nucleic acid sequence of the present invention includes at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the amino acid sequences selected from the group consisting of nucleotides 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 110, 111, 112, 113

[0035] In certain embodiments, the HE variant has the following amino acid substitutions in any one of SEQ ID NOS: 1-5, or a biologically active fragment thereof: S24C, L26G, R28Y, R30H, N32S, K34V, S35L, S36R, V37S, S40R, E42R, G44S, Q46E, T48S, V68T, A70Y, S72A, S78R, K80Q, V116L, L138M, T143N, S159P, F168L, E178D, C180 At least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of S, F182M, N184E, I186M, S188N, S190N, K191N, L192V, G193I, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0036] In certain embodiments, the HE variant has one of the following amino acid substitutions in any one of SEQ ID NOS: 1-5, or a biologically active fragment thereof: S24C, L26R, R28D, R30H, N32A, K34V, S35L, S36R, V37S, S40R, E42R, G44S, Q46E, T48V, V68T, A70Y, S72A, S78R, K80Q, V116L, L138M, T143N, S159P, F168L, E178D, C18 The nucleic acid sequence contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the amino acid sequences selected from the group consisting of 110S, F182V, N184E, I186K, S188R, K189R, K191P, L192V, G193K, Q195G, Q197R, V199R, S201G, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0037] In further embodiments, the HE variant has one of the following amino acid substitutions in any one of SEQ ID NOs: 1-5 or a biologically active fragment thereof: S24C, L26R, R28D, N32A, K34D, S35L, S36R, V37A, S40R, E42R, G44A, Q46E, T48V, V68T, A70Y, S72A, S78R, K80Q, D92G, L138M, T143N, S159P, F168L, E178D, C180S, At least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of F182M, N184E, I186M, S188N, S190N, K191N, L192V, G193I, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0038] In certain embodiments, the HE variant comprises an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 6-12, or a biologically active fragment thereof.

[0039] In some embodiments, the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.

[0040] In additional embodiments, the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 7, or a biologically active fragment thereof.

[0041] In certain embodiments, the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 8, or a biologically active fragment thereof.

[0042] In certain embodiments, the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 9, or a biologically active fragment thereof.

[0043] In a further embodiment, the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 10, or a biologically active fragment thereof.

[0044] In certain embodiments, the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 11, or a biologically active fragment thereof.

[0045] In certain embodiments, the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 12, or a biologically active fragment thereof.

[0046] In some embodiments, the polypeptide binds to the polynucleotide sequence set forth in SEQ ID NO:20.

[0047] In certain embodiments, the polypeptide further comprises a DNA binding domain.

[0048] In additional embodiments, the DNA binding domain is selected from the group consisting of a TALE DNA binding domain and a zinc finger DNA binding domain.

[0049] In certain embodiments, the TALE DNA binding domain comprises between about 9.5 TALE repeat units and about 15.5 TALE repeat units.

[0050] In a further embodiment, the TALE DNA binding domain binds to a polynucleotide sequence within the CBLB gene.

[0051] In a specific embodiment, the TALE DNA binding domain binds to the polynucleotide sequence shown in SEQ ID NO:21.

[0052] In certain embodiments, the polypeptide binds to and cleaves the polynucleotide sequence set forth in SEQ ID NO:22.

[0053] In certain embodiments, the zinc finger DNA binding domain comprises 2, 3, 4, 5, 6, 7, or 8 zinc finger motifs.

[0054] In additional embodiments, the polypeptide further comprises a peptide linker and an endo-processing enzyme or a biologically active fragment thereof.

[0055] In certain embodiments, the polypeptide further comprises a viral self-cleaving 2A peptide and an endo-processing enzyme or a biologically active fragment thereof.

[0056] In further embodiments, the endo-processing enzyme or biologically active fragment thereof has 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease, 5' flap endonuclease, helicase, TdT, or template-independent DNA polymerase activity.

[0057] In some embodiments, the endo-processing enzyme comprises Trex2 or a biologically active fragment thereof.

[0058] In further embodiments, the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 13-19, or a biologically active fragment thereof.

[0059] In additional embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13, or a biologically active fragment thereof.

[0060] In certain embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, or a biologically active fragment thereof.

[0061] In certain embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 15, or a biologically active fragment thereof.

[0062] In certain embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16, or a biologically active fragment thereof.

[0063] In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17, or a biologically active fragment thereof.

[0064] In certain embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18, or a biologically active fragment thereof.

[0065] In a further embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 19, or a biologically active fragment thereof.

[0066] In a specific embodiment, the polypeptide truncates the human CBLB gene at the polynucleotide sequence shown in SEQ ID NO:20 or 22.

[0067] In various embodiments, the present disclosure provides, in part, polynucleotides that encode the polypeptides contemplated herein.

[0068] In some embodiments, the present disclosure provides, in part, mRNAs that encode the polypeptides contemplated herein.

[0069] In certain embodiments, the present disclosure provides, in part, cDNAs encoding the polypeptides contemplated herein.

[0070] In various embodiments, the present disclosure provides, in part, a vector comprising a polynucleotide encoding a polypeptide contemplated herein.

[0071] In various embodiments, the present disclosure provides, in part, cells comprising the polypeptides contemplated herein.

[0072] In additional embodiments, the present disclosure provides, in part, a cell comprising a polynucleotide encoding a polypeptide contemplated herein.

[0073] In a further embodiment, the present disclosure provides, in part, a cell comprising a vector contemplated herein.

[0074] In some embodiments, the present disclosure provides, in part, cells comprising one or more genomic modifications introduced by a polypeptide contemplated herein.

[0075] In certain embodiments, the cells comprise a polynucleotide encoding one or more of an immunopotency enhancer, an immunosuppressive signal damper, or an engineered antigen receptor.

[0076] In a further embodiment, the polynucleotide further comprises an RNA polymerase II promoter operably linked to a polynucleotide encoding an immunopotency enhancer, an immunosuppressive signal damper, or an engineered antigen receptor.

[0077] In some embodiments, the RNA polymerase II promoter is selected from the group consisting of the short EF1α promoter, the long EF1α promoter, the human ROSA 26 locus, the ubiquitin C (UBC) promoter, the phosphoglycerate kinase-1 (PGK) promoter, the cytomegalovirus enhancer / chicken β-actin (CAG) promoter, the β-actin promoter, and the dl587rev primer binding site substitution (MND) promoter of the myeloproliferative sarcoma virus enhancer with deleted negative regulatory regions.

[0078] In additional embodiments, the polynucleotide further encodes one or more self-cleaving viral peptides operably linked to, interspersed between, and / or adjacent to an immune enhancer, an immunosuppressive signal damper, or an engineered antigen receptor.

[0079] In certain embodiments, the self-cleaving viral peptide is a 2A peptide.

[0080] In certain embodiments, the polynucleotide further comprises a heterologous polyadenylation signal.

[0081] In certain embodiments, the immunosuppressive signal damper comprises an enzymatic function that opposes an immunosuppressive factor.

[0082] In additional embodiments, the immunosuppressive signal damper comprises kynureninase activity.

[0083] In certain embodiments, the immunosuppressive signal damper comprises an extracellular domain that binds to an immunosuppressive factor, which extracellular domain is optionally an antibody or an antigen-binding fragment thereof, an extracellular domain that binds to the immunosuppressive factor and a transmembrane domain, or an extracellular domain that binds to the immunosuppressive factor, a transmembrane domain, and a modified intracellular domain that is incapable of transmitting an immunosuppressive signal to a cell.

[0084] In further embodiments, the immune enhancer is selected from the group consisting of a bispecific T cell engager molecule (BiTE), an immunostimulatory factor, and a flip receptor.

[0085] In some embodiments, the immunostimulatory factor is selected from the group consisting of a cytokine, a chemokine, a cytotoxin, a cytokine receptor, and variants thereof.

[0086] In some embodiments, the flip receptor comprises the TGFβRII extracellular domain and transmembrane domain and the intracellular domain from TLR4, CD28, CD134, CD137, CD278, and / or CD3ζ fused in-frame to the C-terminal end of the TGFβRII transmembrane domain.

[0087] In certain embodiments, the flip receptor comprises a TGFβRII extracellular domain, a transmembrane domain isolated from a TLR4, CD3, CD4, CD8α, CD28, CD134, or CD137 polypeptide, and an intracellular domain from TLR4, CD28, CD134, CD137, CD278, and / or CD3ζ fused in-frame to the C-terminal end of the TGFβRII extracellular domain.

[0088] In certain embodiments, the flip receptor comprises a TGFβRII extracellular domain and a transmembrane domain and an intracellular domain isolated from a TLR4, CD3, CD4, CD8α, CD28, CD134, or CD137 polypeptide fused in-frame to the C-terminal end of the TGFβRII extracellular domain.

[0089] In certain embodiments, the engineered antigen receptor is selected from the group consisting of an engineered TCR, CAR, Daric, or zetakine.

[0090] In an additional embodiment, the engineered receptor is not integrated into the CBLB gene.

[0091] In a further embodiment, a polynucleotide encoding one or more of an immunopotency enhancer, an immunosuppressive signal damper, or an engineered antigen receptor is integrated into the CBLB gene.

[0092] In a further embodiment, the donor repair template comprises a polynucleotide encoding one or more of an immune enhancer, an immune suppressive signal damper, or an engineered antigen receptor, and is integrated into the CBLB gene at the site of the DNA double-strand break introduced by the polypeptide contemplated herein.

[0093] In certain embodiments, the cells are hematopoietic cells.

[0094] In some embodiments, the cell is a T cell.

[0095] In certain embodiments, the cells are CD3+, CD4+, and / or CD8+ cells.

[0096] In additional embodiments, the cell is an immune effector cell.

[0097] In certain embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell.

[0098] In certain embodiments, the cells are natural killer (NK) cells or natural killer T (NKT) cells.

[0099] In a preferred embodiment, the cells are T cells that have been genetically modified to express an engineered receptor.

[0100] In a preferred embodiment, the cells are T cells that have been genetically modified to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

[0101] In additional embodiments, the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor.

[0102] In some embodiments, the cells comprise one or more modified CBLB alleles.

[0103] In a further embodiment, one or more modified CBLB alleles are dysfunctional or have substantially reduced CBLB function and / or activity.

[0104] In certain embodiments, the cells comprise a nucleic acid encoding an immune enhancer or an immune suppressive signal damper introduced into one or more modified CBLB alleles, and the cells further comprise an engineered antigen receptor that is not introduced into one or more modified CBLB alleles.

[0105] In some embodiments, the present disclosure provides, in part, a plurality of cells comprising one or more cells contemplated herein.

[0106] In various embodiments, the present disclosure provides, in part, compositions comprising one or more cells contemplated herein.

[0107] In some embodiments, the present disclosure provides, in part, a composition comprising one or more cells contemplated herein and a physiologically acceptable carrier.

[0108] In certain embodiments, the present disclosure provides, in part, a method for editing the human CBLB gene in a cell, comprising introducing into the cell a polynucleotide encoding a polypeptide contemplated herein, wherein expression of the polypeptide creates a double-stranded break at a target site in the human CBLB gene.

[0109] In some embodiments, the disclosure provides, in part, a method of editing the human CBLB gene in a cell, comprising introducing into the cell a polynucleotide encoding a polypeptide contemplated herein, wherein expression of the polypeptide creates a double-stranded break at a target site in the human CBLB gene, and the break is repaired by non-homologous end joining (NHEJ).

[0110] In certain embodiments, the present disclosure provides, in part, a method for editing a human CBLB gene in a cell, comprising introducing into the cell a polynucleotide encoding a polypeptide contemplated herein and a donor repair template, wherein expression of the polypeptide creates a double-stranded break at a target site within the human CBLB gene, and the donor repair template is integrated into the human CBLB gene by homology-directed repair (HDR) at the site of the double-stranded break (DSB).

[0111] In certain embodiments, the cells are hematopoietic cells.

[0112] In certain embodiments, the cell is a T cell.

[0113] In certain embodiments, the cells are CD3+, CD4+, and / or CD8+ cells.

[0114] In some embodiments, the cell is an immune effector cell.

[0115] In additional embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell.

[0116] In additional embodiments, the cells are natural killer (NK) cells or natural killer T (NKT) cells.

[0117] In certain embodiments, the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor.

[0118] In certain embodiments, the polynucleotide encoding the polypeptide is mRNA.

[0119] In certain embodiments, a polynucleotide encoding a 5'-3' exonuclease has been introduced into the cell.

[0120] In a further embodiment, a polynucleotide encoding Trex2 or a biologically active fragment thereof has been introduced into the cell.

[0121] In some embodiments, the donor repair template encodes a CBLB gene or a portion thereof that contains one or more mutations compared to a wild-type CBLB gene.

[0122] In certain embodiments, the donor repair template encodes one or more of an immune enhancer, an immune suppressive signal damper, or an engineered antigen receptor.

[0123] In some embodiments, the donor repair template further comprises an RNA polymerase II promoter operably linked to an immunopotency enhancer, an immunosuppressive signal damper, or an engineered antigen receptor.

[0124] In further embodiments, the RNA polymerase II promoter is selected from the group consisting of a short EF1α promoter, a long EF1α promoter, a human ROSA26 locus, a ubiquitin C (UBC) promoter, a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus enhancer / chicken β-actin (CAG) promoter, a β-actin promoter, and a myeloproliferative sarcoma virus enhancer, a deleted negative regulatory region, a dl587rev primer binding site substitution (MND) promoter.

[0125] In certain embodiments, the donor repair template further encodes one or more self-cleaving viral peptides operably linked to, interspersed between, and / or adjacent to an immune enhancer, an immune suppressive signal damper, or an engineered antigen receptor.

[0126] In an additional embodiment, the self-cleaving viral peptide is a 2A peptide.

[0127] In certain embodiments, the donor repair template further comprises a heterologous polyadenylation signal.

[0128] In a further embodiment, the immunosuppressive signal damper comprises an enzymatic function that opposes the immunosuppressive factor.

[0129] In certain embodiments, the immunosuppressive signal damper comprises kynureninase activity.

[0130] In some embodiments, the immunosuppressive signal damper comprises an extracellular domain that binds to an immunosuppressive factor, which extracellular domain is optionally an antibody or an antigen-binding fragment thereof, an extracellular domain that binds to the immunosuppressive factor and a transmembrane domain, or an extracellular domain that binds to the immunosuppressive factor, a transmembrane domain, and a modified intracellular domain that is incapable of transmitting an immunosuppressive signal to a cell.

[0131] In certain embodiments, the extracellular and / or transmembrane domain of the immunosuppressive signal damper is a TGFβRII extracellular and / or transmembrane domain.

[0132] In some embodiments, the immune enhancer is selected from the group consisting of a bispecific T cell engager molecule (BiTE), an immunostimulatory factor, and a flip receptor.

[0133] In certain embodiments, the immunostimulatory factor is selected from the group consisting of a cytokine, a chemokine, a cytotoxin, a cytokine receptor, and variants thereof.

[0134] In additional embodiments, the flip receptor comprises the TGFβRII extracellular domain and transmembrane domain and the intracellular domain from TLR4, CD28, CD134, CD137, CD278, and / or CD3ζ fused in-frame to the C-terminal end of the TGFβRII transmembrane domain.

[0135] In certain embodiments, the flip receptor comprises a TGFβRII extracellular domain, a transmembrane domain isolated from a TLR4, CD3, CD4, CD8α, CD28, CD134, or CD137 polypeptide, and an intracellular domain from TLR4, CD28, CD134, CD137, CD278, and / or CD3ζ fused in-frame to the C-terminal end of the TGFβRII extracellular domain.

[0136] In a further embodiment, the flip receptor comprises a TGFβRII extracellular domain and a transmembrane domain and an intracellular domain isolated from a TLR4, CD3, CD4, CD8α, CD28, CD134, or CD137 polypeptide fused in-frame to the C-terminal end of the TGFβRII extracellular domain.

[0137] In certain embodiments, the engineered antigen receptor is selected from the group consisting of an engineered TCR, CAR, Daric, or zetakine.

[0138] In some embodiments, the donor repair template comprises a 5' homology arm that is homologous to the human CBLB gene sequence 5' of the DSB and a 3' homology arm that is homologous to the human CBLB gene sequence 3' of the DSB.

[0139] In a further embodiment, the lengths of the 5' and 3' homology arms are independently selected from about 100 bp to about 2500 bp.

[0140] In certain embodiments, the lengths of the 5' and 3' homology arms are independently selected from about 600 bp to about 1500 bp.

[0141] In a specific embodiment, the 5' homology arm is about 1500 bp and the 3' homology arm is about 1000 bp.

[0142] In some embodiments, the 5' homology arm is about 600 bp and the 3' homology arm is about 600 bp.

[0143] In certain embodiments, a viral vector is used to introduce the donor repair template into the cell.

[0144] In additional embodiments, the viral vector is a recombinant adeno-associated viral vector (rAAV) or a retrovirus.

[0145] In certain embodiments, the rAAV has one or more ITRs from AAV2.

[0146] In additional embodiments, the rAAV has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.

[0147] In a further embodiment, the rAAV has an AAV2 or AAV6 serotype.

[0148] In certain embodiments, the retrovirus is a lentivirus.

[0149] In some embodiments, the lentivirus is an integrase-deficient lentivirus (IDLV).

[0150] In various embodiments, the present disclosure provides, in part, methods for treating, preventing, or alleviating at least one symptom of cancer, infectious disease, autoimmune disease, inflammatory disease, and immune deficiency, or a condition related thereto, comprising administering to a subject an effective amount of a composition contemplated herein.

[0151] In certain embodiments, the present disclosure provides, in part, a method of treating a solid tumor, comprising administering to a subject an effective amount of a composition contemplated herein.

[0152] In certain embodiments, solid cancers include liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.

[0153] In some embodiments, the present disclosure provides, in part, a method of treating a hematological malignancy comprising administering to a subject an effective amount of a composition contemplated herein.

[0154] In certain embodiments, the hematological malignancy is leukemia, lymphoma, or multiple myeloma. The present invention provides, for example, the following items. (Item 1) A polypeptide comprising a homing endonuclease (HE) variant that cleaves a target site in the human casitas lineage B (Cbl) lymphoma proto-oncogene B (CBLB) gene. (Item 2) 2. The polypeptide of item 1, wherein the HE variant is a LAGLIDADG homing endonuclease (LHE) variant. (Item 3) Item 3. The polypeptide of item 1 or 2, wherein the polypeptide comprises a biologically active fragment of the HE variant. (Item 4) 4. The polypeptide of item 3, wherein the biologically active fragment lacks 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal amino acids compared to the corresponding wild-type HE. (Item 5) 5. The polypeptide according to item 4, wherein the biologically active fragment lacks four N-terminal amino acids compared to the corresponding wild-type HE. (Item 6) 5. The polypeptide according to item 4, wherein the biologically active fragment lacks 8 N-terminal amino acids compared to the corresponding wild-type HE. (Item 7) 4. The polypeptide of item 3, wherein the biologically active fragment lacks 1, 2, 3, 4, 5, or 6 C-terminal amino acids compared to the corresponding wild-type HE. (Item 8) 8. The polypeptide of item 7, wherein the biologically active fragment lacks a C-terminal amino acid compared to the corresponding wild-type HE. (Item 9) 8. The polypeptide of item 7, wherein the biologically active fragment lacks two C-terminal amino acids compared to the corresponding wild-type HE. (Item 10) The HE variant is I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I -CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I 10. The polypeptide of any one of items 1 to 9, wherein the LHE variant is selected from the group consisting of I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, and I-Vdi141I. (Item 11) 11. The polypeptide of any one of items 1 to 10, wherein the HE variant is a variant of LHE selected from the group consisting of I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, and SmaMI. (Item 12) 12. The polypeptide of any one of items 1 to 11, wherein the HE variant is an I-OnuI LHE variant. (Item 13) 13. The polypeptide of any one of items 1 to 12, wherein the HE variant comprises one or more amino acid substitutions in the DNA recognition interface at amino acid positions selected from the group consisting of 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76, 78, 80, 82, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of the I-OnuI LHE amino acid sequence set forth in SEQ ID NOs: 1 to 5, or a biologically active fragment thereof. (Item 14) The HE variants have at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions in I-OnuI as shown in SEQ ID NOs: 1 to 5. 14. The polypeptide of any one of items 1 to 13, comprising an LHE amino acid sequence or a biologically active fragment thereof at amino acid positions selected from the group consisting of 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76, 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240. (Item 15) The HE variant has at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions at positions 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 74, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 142, 144, 146, 148, 150, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 15. The polypeptide of any one of items 1 to 14, comprising at least one position selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 14, 15, 16, 17, 18, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 207, 223, 225, 227, 232, 236, and 238. (Item 16) 10. The HE variant comprises any one of the following amino acid substitutions in any one of SEQ ID NOS: 1 to 5 or a biologically active fragment thereof: S24C, L26R, L26G, R28D, R28Y, R30H, N32A, N32S, K34D, K34V, S35L, S36R, V37A, V37S, S40R, E42R, G44A, G44S, Q46E, T48V, T48S, V68T, V68K, A70Y, S72A, S78R, K80Q, D92G, V116L, L138M, T143N, S159P, F168L, E178D, C180S, F182V, F182M, N184 16. The polypeptide of any one of items 1 to 15, comprising at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acids: E, I186K, I186M, S188R, S188N, K189R, S190N, K191P, K191N, L192V, G193K, G193I, Q195G, Q195R, Q197R, V199R, S201G, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I. (Item 17) 10. The HE variant comprises any one of the following amino acid substitutions in any one of SEQ ID NOS: 1 to 5, or a biologically active fragment thereof: S24C, L26R, R28D, N32A, K34D, S35L, S36R, V37A, S40R, E42R, G44A, Q46E, T48V, V68T, A70Y, S72A, S78R, K80Q, L138M, T143N, F168L, E178D, C180S, F182V, N184E, I186K, S1 17. The polypeptide of any one of items 1 to 16, comprising at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of 88R, K189R, K191P, L192V, G193K, Q195G, Q197R, V199R, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I. (Item 18) 10. The HE variant comprises any one of the following amino acid substitutions in any one of SEQ ID NOS: 1 to 5 or a biologically active fragment thereof: S24C, L26R, R28D, N32A, K34D, S35L, S36R, V37A, S40R, E42R, G44A, Q46E, T48V, V68T, A70Y, S72A, S78R, K80Q, L138M, T143N, S159P, F168L, E178D, C180S, F182M, N184E, I186M, S1 17. The polypeptide of any one of items 1 to 16, comprising at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of 88N, S190N, K191N, L192V, G193I, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I. (Item 19) 10. The HE variant comprises any one of the following amino acid substitutions in any one of SEQ ID NOS: 1 to 5, or a biologically active fragment thereof: S24C, L26R, R28D, N32A, K34D, S35L, S36R, V37A, S40R, E42R, G44S, Q46E, T48S, V68T, A70Y, S72A, S78R, K80Q, D92G, V116L, L138M, T143N, S159P, F168L, E178D, C180S, F182M, N184E, I18 17. The polypeptide of any one of items 1 to 16, comprising at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of 6M, S188N, S190N, K191N, L192V, G193I, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I. (Item 20) 10. The HE variant comprises any one of the following amino acid substitutions in any one of SEQ ID NOS: 1 to 5, or a biologically active fragment thereof: S24C, L26R, R28D, R30H, N32A, K34V, S35L, S36R, V37S, S40R, E42R, G44S, Q46E, T48V, V68T, V68K, A70Y, S72A, S78R, K80Q, L138M, T143N, S159P, F168L, E178D, C180S, F182M, N184E, I18 17. The polypeptide of any one of items 1 to 16, comprising at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of 6M, S188N, S190N, K191N, L192V, G193I, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I. (Item 21) 10. The HE variant comprises any one of the following amino acid substitutions in any one of SEQ ID NOS: 1 to 5, or a biologically active fragment thereof: S24C, L26G, R28Y, R30H, N32S, K34V, S35L, S36R, V37S, S40R, E42R, G44S, Q46E, T48S, V68T, A70Y, S72A, S78R, K80Q, V116L, L138M, T143N, S159P, F168L, E178D, C180S, F182M, N184E, I18 17. The polypeptide of any one of items 1 to 16, comprising at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of 6M, S188N, S190N, K191N, L192V, G193I, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I. (Item 22) 10. The HE variant comprises any one of the following amino acid substitutions in any one of SEQ ID NOS: 1 to 5, or a biologically active fragment thereof: S24C, L26R, R28D, R30H, N32A, K34V, S35L, S36R, V37S, S40R, E42R, G44S, Q46E, T48V, V68T, A70Y, S72A, S78R, K80Q, V116L, L138M, T143N, S159P, F168L, E178D, C180S, F182V, N184E, I18 17. The polypeptide of any one of items 1 to 16, comprising at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of 6K, S188R, K189R, K191P, L192V, G193K, Q195G, Q197R, V199R, S201G, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I. (Item 23) The HE variant comprises any one of the following amino acid substitutions in any one of SEQ ID NOs: 1 to 5 or a biologically active fragment thereof: S24C, L26R, R28D, N32A, K34D, S35L, S36R, V37A, S40R, E42R, G44A, Q46E, T48V, V68T, A70Y, S72A, S78R, K80Q, D92G, L138M, T143N, S159P, F168L, E178D, C180S, F182M, N184E, I186M, 17. The polypeptide of any one of items 1 to 16, comprising at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acids: S188N, S190N, K191N, L192V, G193I, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I. (Item 24) 24. The polypeptide according to any one of items 1 to 23, wherein the HE variant comprises an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 6 to 12, or a biologically active fragment thereof. (Item 25) 25. The polypeptide of any one of items 1 to 24, wherein the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 6 or a biologically active fragment thereof. (Item 26) 25. The polypeptide of any one of items 1 to 24, wherein the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 7 or a biologically active fragment thereof. (Item 27) 25. The polypeptide of any one of items 1 to 24, wherein the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 8 or a biologically active fragment thereof. (Item 28) 25. The polypeptide of any one of items 1 to 24, wherein the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 9 or a biologically active fragment thereof. (Item 29) 25. The polypeptide of any one of items 1 to 24, wherein the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 10 or a biologically active fragment thereof. (Item 30) 25. The polypeptide of any one of items 1 to 24, wherein the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 11 or a biologically active fragment thereof. (Item 31) 25. The polypeptide of any one of items 1 to 24, wherein the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 12 or a biologically active fragment thereof. (Item 32) 32. The polypeptide according to any one of items 1 to 31, wherein the polypeptide binds to the polynucleotide sequence shown in SEQ ID NO: 20. (Item 33) 33. The polypeptide of any one of items 1 to 32, further comprising a DNA-binding domain. (Item 34) 34. The polypeptide of item 33, wherein the DNA binding domain is selected from the group consisting of a TALE DNA binding domain and a zinc finger DNA binding domain. (Item 35) 35. The polypeptide of item 34, wherein the TALE DNA binding domain comprises about 9.5 TALE repeat units to about 15.5 TALE repeat units. (Item 36) The polypeptide of item 34 or item 35, wherein the TALE DNA binding domain binds to a polynucleotide sequence within the CBLB gene. (Item 37) 37. The polypeptide of any one of items 34 to 36, wherein the TALE DNA binding domain binds to the polynucleotide sequence shown in SEQ ID NO: 21. (Item 38) 38. The polypeptide according to item 37, wherein the polypeptide binds to and cleaves the polynucleotide sequence set forth in SEQ ID NO: 22. (Item 39) 35. The polypeptide of item 34, wherein the zinc finger DNA-binding domain comprises 2, 3, 4, 5, 6, 7, or 8 zinc finger motifs. (Item 40) 40. The polypeptide of any one of items 1 to 39, further comprising a peptide linker and an endo-processing enzyme or a biologically active fragment thereof. (Item 41) 41. The polypeptide of any one of items 1 to 40, further comprising a viral self-cleaving 2A peptide and an endo-processing enzyme or a biologically active fragment thereof. (Item 42) 42. The polypeptide of claim 40 or 41, wherein the endo-processing enzyme or biologically active fragment thereof has 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease, 5' flap endonuclease, helicase, TdT, or template-independent DNA polymerase activity. (Item 43) 43. The polypeptide according to any one of items 40 to 42, wherein the endo-processing enzyme comprises Trex2 or a biologically active fragment thereof. (Item 44) 44. The polypeptide according to any one of items 1 to 43, wherein the polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13 to 19 or a biologically active fragment thereof. (Item 45) 45. The polypeptide according to item 44, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13 or a biologically active fragment thereof. (Item 46) 45. The polypeptide according to item 44, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14 or a biologically active fragment thereof. (Item 47) 45. The polypeptide according to item 44, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 15 or a biologically active fragment thereof. (Item 48) 45. The polypeptide according to item 44, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16 or a biologically active fragment thereof. (Item 49) 45. The polypeptide according to item 44, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17 or a biologically active fragment thereof. (Item 50) 45. The polypeptide according to item 44, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18 or a biologically active fragment thereof. (Item 51) 45. The polypeptide according to item 44, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 19 or a biologically active fragment thereof. (Item 52) 52. The polypeptide of any one of items 1 to 51, wherein the polypeptide cleaves the human CBLB gene at the polynucleotide sequence shown in SEQ ID NO: 20 or 22. (Item 53) 53. A polynucleotide encoding the polypeptide according to any one of items 1 to 52. (Item 54) 53. An mRNA encoding the polypeptide according to any one of items 1 to 52. (Item 55) A cDNA encoding the polypeptide according to any one of items 1 to 52. (Item 56) 53. A vector comprising a polynucleotide encoding the polypeptide according to any one of items 1 to 52. (Item 57) 53. A cell comprising the polypeptide according to any one of items 1 to 52. (Item 58) 53. A cell comprising a polynucleotide encoding the polypeptide of any one of items 1 to 52. (Item 59) A cell containing the vector described in item 56. (Item 60) 53. A cell comprising one or more genomic modifications introduced by the polypeptide of any one of items 1 to 52. (Item 61) 61. The cell of any one of items 57 to 60, wherein the cell comprises a polynucleotide encoding one or more of an immune enhancer, an immune suppressive signal damper, or an engineered antigen receptor. (Item 62) 62. The cell of item 61, wherein the polynucleotide further comprises an RNA polymerase II promoter operably linked to the polynucleotide encoding the immune enhancer, immune suppressive signal damper, or engineered antigen receptor. (Item 63) 63. The cell of item 62, wherein the RNA polymerase II promoter is selected from the group consisting of a short EF1α promoter, a long EF1α promoter, a human ROSA 26 locus, a ubiquitin C (UBC) promoter, a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus enhancer / chicken β-actin (CAG) promoter, a β-actin promoter, and a dl587rev primer binding site substitution (MND) promoter of the myeloproliferative sarcoma virus enhancer with deleted negative regulatory regions. (Item 64) 64. The cell of any one of items 61 to 63, wherein the polynucleotide further encodes one or more self-cleaving viral peptides operably linked to, interspersed between, and / or adjacent to the immune enhancer, immune suppressive signal damper, or engineered antigen receptor. (Item 65) Item 66. The cell according to Item 64, wherein the self-cleaving viral peptide is a 2A peptide. 66. The cell of any one of items 61 to 65, wherein the polynucleotide further comprises a heterologous polyadenylation signal. (Item 67) 67. The cell according to any one of items 61 to 66, wherein the immunosuppressive signal damper comprises an enzymatic function that opposes an immunosuppressive factor. (Item 68) 68. The cell of item 67, wherein the immunosuppressive signal damper comprises kynureninase activity. (Item 69) The immunosuppressive signal damper (a) an extracellular domain that binds to an immunosuppressive factor, optionally an antibody or antigen-binding fragment thereof; (b) an extracellular domain and a transmembrane domain that binds to an immunosuppressive factor, or (c) The cell according to any one of items 61 to 66, comprising an extracellular domain that binds to an immunosuppressive factor, a transmembrane domain, and a modified intracellular domain that is incapable of transmitting an immunosuppressive signal to said cell. (Item 70) 67. The cell of any one of items 61 to 66, wherein the immune enhancer is selected from the group consisting of a bispecific T cell engager molecule (BiTE), an immunostimulatory factor, and a flip receptor. (Item 71) 71. The cell of item 70, wherein the immunostimulatory factor is selected from the group consisting of cytokines, chemokines, cytotoxins, cytokine receptors, and variants thereof. (Item 72) 71. The cell of item 70, wherein the flip receptor comprises a TGFβRII extracellular domain and transmembrane domain and an intracellular domain from TLR4, CD28, CD134, CD137, CD278, and / or CD3ζ fused in-frame to the C-terminal end of the TGFβRII transmembrane domain. (Item 73) 71. The cell of paragraph 70, wherein the flip receptor comprises a TGFβRII extracellular domain, a transmembrane domain isolated from a TLR4, CD3, CD4, CD8α, CD28, CD134, or CD137 polypeptide, and an intracellular domain from TLR4, CD28, CD134, CD137, CD278, and / or CD3ζ fused in-frame to the C-terminal end of the TGFβRII extracellular domain. (Item 74) 71. The cell of item 70, wherein the flip receptor comprises a TGFβRII extracellular domain and a transmembrane domain and an intracellular domain isolated from a TLR4, CD3, CD4, CD8α, CD28, CD134, or CD137 polypeptide fused in-frame to the C-terminal end of the TGFβRII extracellular domain. (Item 75) 67. The cell of any one of items 61 to 66, wherein the engineered antigen receptor is selected from the group consisting of an engineered TCR, CAR, Daric, or zetakine. (Item 76) 76. The cell of item 75, wherein the engineered receptor is not integrated into the CBLB gene. (Item 77) 67. The cell of any one of items 61 to 66, wherein the polynucleotide encoding one or more of an immune enhancer, an immune suppressive signal damper, or an engineered antigen receptor is integrated into the CBLB gene. (Item 78) 67. The cell of any one of items 61 to 66, wherein a donor repair template comprising the polynucleotide encoding one or more of an immune enhancer, an immune suppressive signal damper, or a genetically engineered antigen receptor is integrated into the CBLB gene at the site of the DNA double-strand break introduced by the polypeptide of any one of items 1 to 52. (Item 79) 79. The cell according to any one of items 57 to 78, wherein the cell is a hematopoietic cell. (Item 80) 80. The cell according to any one of items 57 to 79, wherein the cell is a T cell. (Item 81) The cells are CD3 + , CD4 + , and / or CD8 + 81. The cell according to any one of items 57 to 80, which is a cell. (Item 82) 82. The cell according to any one of items 57 to 81, wherein the cell is an immune effector cell. (Item 83) 83. The cell of any one of items 57 to 82, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. (Item 84) 83. The cell of any one of items 57 to 82, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. (Item 85) 85. The cell according to any one of items 57 to 84, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, or a tumor. (Item 86) 86. The cell of any one of items 47 to 85, wherein the cell comprises one or more modified CBLB alleles. (Item 87) 73. The cell of item 72, wherein the one or more modified CBLB alleles are dysfunctional or have substantially reduced CBLB function and / or activity. (Item 88) 52. The cell of any one of items 46 to 51, wherein the cell comprises a nucleic acid encoding an immune enhancer or an immune suppressive signal damper introduced into the one or more modified CBLB alleles, and the cell further comprises an engineered antigen receptor not introduced into the one or more modified CBLB alleles. (Item 89) 89. A plurality of cells comprising one or more cells according to any one of items 57 to 88. (Item 90) 89. A composition comprising one or more cells according to any one of items 57 to 88. (Item 91) 89. A composition comprising one or more cells according to any one of items 57 to 88 and a physiologically acceptable carrier. (Item 92) 53. A method for editing the human CBLB gene in a cell, comprising introducing into said cell a polynucleotide encoding a polypeptide according to any one of items 1 to 52, wherein expression of said polypeptide creates a double-stranded break at a target site in the human CBLB gene. (Item 93) 53. A method for editing the human CBLB gene in a cell, comprising introducing into said cell a polynucleotide encoding a polypeptide according to any one of items 1 to 52, wherein expression of said polypeptide creates a double-stranded break at a target site in the human CBLB gene, and said break is repaired by non-homologous end joining (NHEJ). (Item 94) 53. A method for editing the human CBLB gene in a cell, comprising introducing into said cell a polynucleotide encoding a polypeptide according to any one of items 1 to 52 and a donor repair template, wherein expression of said polypeptide creates a double-strand break at a target site in the human CBLB gene, and wherein said donor repair template is integrated into said human CBLB gene by homology-directed repair (HDR) at the site of the double-strand break (DSB). (Item 95) 95. The method of any one of items 92 to 94, wherein the cells are hematopoietic cells. (Item 96) 96. The method of any one of items 92 to 95, wherein the cells are T cells. (Item 97) The cells are CD3 + , CD4 + , and / or CD8 + 97. The method of any one of items 92 to 96, wherein the cell is a cell. (Item 98) 98. The method of any one of items 92 to 97, wherein the cells are immune effector cells. (Item 99) 99. The method of any one of items 92 to 98, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. (Item 100) 99. The method of any one of items 92 to 98, wherein the cells are natural killer (NK) cells or natural killer T (NKT) cells. (Item 101) 101. The method of any one of items 92 to 100, wherein the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, or a tumor. (Item 102) 102. The method of any one of items 92 to 101, wherein the polynucleotide encoding the polypeptide is mRNA. (Item 103) 103. The method of any one of items 92 to 102, wherein a polynucleotide encoding a 5'-3' exonuclease is introduced into the cell. (Item 104) 104. The method of any one of items 92 to 103, wherein a polynucleotide encoding Trex2 or a biologically active fragment thereof is introduced into the cell. (Item 105) 105. The method of any one of items 94 to 104, wherein the donor repair template encodes a CBLB gene or a portion thereof comprising one or more mutations compared to the wild-type CBLB gene. (Item 106) 106. The method of any one of items 94 to 105, wherein the donor repair template encodes one or more of an immune enhancer, an immune suppressive signal damper, or an engineered antigen receptor. (Item 107) 107. The method of claim 106, wherein the donor repair template further comprises an RNA polymerase II promoter operably linked to the immune enhancer, immune suppressive signal damper, or engineered antigen receptor. (Item 108) 108. The method of claim 107, wherein the RNA polymerase II promoter is selected from the group consisting of a short EF1α promoter, a long EF1α promoter, a human ROSA 26 locus, a ubiquitin C (UBC) promoter, a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus enhancer / chicken β-actin (CAG) promoter, a β-actin promoter, and a dl587rev primer binding site substitution (MND) promoter of the myeloproliferative sarcoma virus enhancer with deleted negative regulatory regions. (Item 109) 109. The method of any one of items 106 to 108, wherein the donor repair template further encodes one or more self-cleaving viral peptides operably linked to, interspersed between, and / or adjacent to the immune enhancer, immune suppressive signal damper, or engineered antigen receptor. (Item 110) Item 111. The method of item 109, wherein the self-cleaving viral peptide is a 2A peptide. 109. The method of any one of items 106 to 108, wherein the donor repair template further comprises a heterologous polyadenylation signal. (Item 112) 112. The method according to any one of items 106 to 111, wherein the immunosuppressive signal damper comprises an enzymatic function that opposes an immunosuppressive factor. (Item 113) 113. The method of claim 112, wherein the immunosuppressive signal dampener comprises kynureninase activity. (Item 114) The immunosuppressive signal damper (a) an extracellular domain that binds to an immunosuppressive factor, optionally an antibody or antigen-binding fragment thereof; (b) an extracellular domain and a transmembrane domain that binds to an immunosuppressive factor, or (c) The method according to any one of Items 106 to 111, comprising an extracellular domain that binds to an immunosuppressive factor, a transmembrane domain, and a modified intracellular domain that is incapable of transmitting an immunosuppressive signal to said cell. (Item 115) 115. The method of claim 114, wherein the extracellular domain and / or transmembrane domain of the immunosuppressive signal damper is the TGFβRII extracellular domain and / or transmembrane domain. (Item 116) 112. The method of any one of items 106 to 111, wherein the immune enhancer is selected from the group consisting of a bispecific T cell engager molecule (BiTE), an immunostimulatory factor, and a flip receptor. (Item 117) 117. The method of claim 116, wherein the immunostimulatory factor is selected from the group consisting of cytokines, chemokines, cytotoxins, cytokine receptors, and variants thereof. (Item 118) 117. The method of claim 116, wherein the flip receptor comprises a TGFβRII extracellular domain and transmembrane domain and an intracellular domain from TLR4, CD28, CD134, CD137, CD278, and / or CD3ζ fused in-frame to the C-terminal end of the TGFβRII transmembrane domain. (Item 119) 117. The method of claim 116, wherein the flip receptor comprises a TGFβRII extracellular domain, a transmembrane domain isolated from a TLR4, CD3, CD4, CD8α, CD28, CD134, or CD137 polypeptide, and an intracellular domain from TLR4, CD28, CD134, CD137, CD278, and / or CD3ζ fused in-frame to the C-terminal end of the TGFβRII extracellular domain. (Item 120) 117. The method of claim 116, wherein the flip receptor comprises a TGFβRII extracellular domain and a transmembrane domain and an intracellular domain isolated from a TLR4, CD3, CD4, CD8α, CD28, CD134, or CD137 polypeptide fused in-frame to the C-terminal end of the TGFβRII extracellular domain. (Item 121) 112. The method of any one of items 106 to 111, wherein the engineered antigen receptor is selected from the group consisting of an engineered TCR, CAR, Daric, or zetakine. (Item 122) 122. The method of any one of items 94 to 121, wherein the donor repair template comprises a 5' homology arm homologous to the 5' human CBLB gene sequence of the DSB and a 3' homology arm homologous to the 3' human CBLB gene sequence of the DSB. (Item 123) 123. The method of item 122, wherein the lengths of the 5' and 3' homology arms are independently selected from about 100 bp to about 2500 bp. (Item 124) Item 124. The method of item 122 or item 123, wherein the lengths of the 5' and 3' homology arms are independently selected from about 600 bp to about 1500 bp. (Item 125) 125. The method of any one of items 122 to 124, wherein the 5' homology arm is about 1500 bp and the 3' homology arm is about 1000 bp. (Item 126) 126. The method of any one of items 122 to 125, wherein the 5' homology arm is about 600 bp and the 3' homology arm is about 600 bp. (Item 127) 127. The method of any one of items 94 to 126, wherein a viral vector is used to introduce the donor repair template into the cell. (Item 128) 128. The method of claim 127, wherein the viral vector is a recombinant adeno-associated viral vector (rAAV) or a retrovirus. (Item 129) 129. The method of claim 128, wherein the rAAV has one or more ITRs from AAV2. (Item 130) 130. The method of claim 128 or 129, wherein the rAAV has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. (Item 131) 131. The method of any one of items 128 to 130, wherein the rAAV has an AAV2 or AAV6 serotype. (Item 132) 129. The method of claim 128, wherein the retrovirus is a lentivirus. (Item 133) 133. The method of claim 132, wherein the lentivirus is an integrase-deficient lentivirus (IDLV). (Item 134) 92. A method for treating, preventing, or alleviating at least one symptom of cancer, infectious disease, autoimmune disease, inflammatory disease, and immune deficiency, or a condition related thereto, comprising administering to a subject an effective amount of the composition described in item 90 or item 91. (Item 135) 92. A method for treating a solid tumor, comprising administering to the subject an effective amount of the composition according to item 90 or item 91. (Item 136) 136. The method of item 135, wherein the solid cancer comprises liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer. (Item 137) 92. A method for treating hematological malignancies, comprising administering to said subject an effective amount of the composition of item 90 or item 91. (Item 138) 138. The method of claim 137, wherein the hematological malignancy is leukemia, lymphoma, or multiple myeloma. [Brief explanation of the drawings]

[0155] [Figure 1] The CBLB gene and the HE target site in exon 6, which encodes the SH2 domain (SEQ ID NOs: 20 and 76), are shown. [Figure 2] Through three rounds of selection, we show how CBLB HE were reprogrammed via genetic engineering of the NTD and CTD to chimeric "half-sites," followed by fusion of the reprogrammed domains and screening against the complete CBLB target site to isolate fully reprogrammed HE. [Figure 3] 1 shows the activity of CBLB HE variants in a chromosomal reporter assay. [Figure 4] 1 shows yeast surface affinity titration of CBLB.E3 HE variants. [Figure 5-1] Shown is the alignment of the CBLB HE variants (SEQ ID NOs: 77-83) to the wild-type I-OnuI protein (SEQ ID NO: 1), highlighting non-identical positions. [Figure 5-2] Shown is the alignment of the CBLB HE variants (SEQ ID NOs: 77-83) to the wild-type I-OnuI protein (SEQ ID NO: 1), highlighting non-identical positions. [Figure 5-3] Shown is the alignment of the CBLB HE variants (SEQ ID NOs: 77-83) to the wild-type I-OnuI protein (SEQ ID NO: 1), highlighting non-identical positions. [Figure 6] The binding sites of the TAL RVDs (SEQ ID NOs: 22 and 84) fused to the CBLB.E3 HE variant to generate the CBLB.E3 megaTAL are shown. [Figure 7] Figure 1 shows consistent CBLB gene editing rates in CAR T cells using CBLB megaTALs. [Figure 8] 1 shows reduced intracellular CBLB protein expression in T cells treated with CBLB megaTAL. [Figure 9] We show that CBL-edited T cells produce more IFNγ and TNFα when stimulated with anti-CD3 antibodies or with CAR-specific antigens. [Figure 10]TIDE analysis of T cells edited by co-delivery of various CBLB megaTALs and mRNA encoding TREX2. Editing rates at the targeted locus ranged from 61 to 93%. [Figure 11] A diagram of the HDR strategy for inserting the MND-GFP expression cassette into the CBLB target site using a CBLB megaTAL and an AAV donor is shown. [Figure 12] Figure 11 shows a stable increase in GFP expression in T cells edited using the HDR strategy compared to mimic-edited T cells. [Figure 13] Results of an in vivo mouse study are shown. Mice bearing A549 tumors were treated with vehicle-treated T cells (upper left panel), CBLB megaTAL-edited T cells (upper right panel), anti-EGFR CAR T cells (lower left panel), and CBLB megaTAL-edited anti-EGFR CAR T cells (lower right panel). Mean tumor volume was measured over time. DETAILED DESCRIPTION OF THE INVENTION

[0156] Brief description of sequence identifiers SEQ ID NO: 1 is the amino acid sequence of the wild-type I-OnuI LAGLIDADG homing endonuclease (LHE). SEQ ID NO: 2 is the amino acid sequence of wild-type I-OnuI LHE. SEQ ID NO: 3 is the amino acid sequence of a biologically active fragment of wild-type I-OnuI LHE. SEQ ID NO: 4 is the amino acid sequence of a biologically active fragment of wild-type I-OnuI LHE. SEQ ID NO: 5 is the amino acid sequence of a biologically active fragment of wild-type I-OnuI LHE. SEQ ID NOs: 6-12 show the amino acid sequences of I-OnuI LHE variants reprogrammed to bind to and cleave a target site in the human CBLB gene. SEQ ID NOs: 13 to 19 show the amino acid sequences of I-OnuI LHE variants reprogrammed to bind to and cleave a target site in the human CBLB gene. SEQ ID NO: 20 is the I-OnuI LHE variant target site in exon 6 of the human CBLB gene. SEQ ID NO: 21 is the TALE DNA binding domain target site in exon 6 of the human CBLB gene. SEQ ID NO: 22 is the megaTAL target site in exon 6 of the human CBLB gene. SEQ ID NOs: 23, 25, 27, and 29 show the I-OnuI LHE variant N-terminal domain target site in exon 6 of the human CBLB gene. SEQ ID NOs: 24, 26, and 28 show the I-OnuI LHE variant C-terminal domain target site in exon 6 of the human CBLB gene. SEQ ID NO:30 is the polynucleotide sequence of the CBLB.E3 surface display plasmid. SEQ ID NOs: 31 to 36 show the mRNA sequences encoding CBLB megaTAL. SEQ ID NO: 37 is the mRNA sequence encoding mouse Trex2. SEQ ID NO: 38 is the amino acid sequence encoding mouse Trex2. SEQ ID NOs: 39 to 49 show the amino acid sequences of various linkers. SEQ ID NOs: 50 to 74 show the amino acid sequences of the protease cleavage site and the self-cleaving polypeptide cleavage site. In the above sequences, X, if present, refers to any amino acid, or refers to the absence of an amino acid.

[0157] A. Overview The present disclosure generally relates in part to improved genome editing compositions and methods of using them. Without wishing to be bound by any particular theory, the genome editing compositions contemplated in various embodiments can be used to prevent or treat cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immune deficiencies, or conditions related thereto, or to alleviate at least one symptom thereof. A limitation or problem plaguing existing adoptive cell therapy is the low responsiveness of immune effector cells due to exhaustion mediated by the tumor microenvironment. Exhausted T cells have unique molecular signatures that are significantly different from naive, effector, or memory T cells. Exhausted T cells are T cells with reduced cytokine expression and effector function.

[0158] The Casitas B lineage (Cbl) lymphoma proto-oncogene B (CBLB) is a member of the RING finger family or E3 ubiquitin family and is expressed in a wide range of tissues and cell types, including cells of the hematopoietic system. CBLB promotes the ubiquitination of target substrate proteins through the recruitment of the E2 ubiquitin complex, which conjugates the enzyme to its RING finger domain. CBLB substrate protein ubiquitination can promote proteolysis or interference with protein-protein interactions. CBLB binds to substrate proteins through its SH2-containing tyrosine kinase-binding domain, its proline-rich sequence that interacts with SH3 domain-containing proteins, or its ubiquitin-associated domain that interacts with ubiquitin-tagged proteins.

[0159] CBLB is also involved in the negative regulation of effector T cell activity and persistence. CBLB knockout mouse T cells are hyperproliferative, produce elevated levels of IL2 and IFNγ in response to antigen stimulation, are resistant to TGFβ-mediated suppression, and have a lower activation threshold, indicating that CBLB plays a role in negatively regulating T cell activation. Without wishing to be bound by any particular theory, it is contemplated that disruption of the CBLB gene in T cells using genetically engineered nucleases will result in more effective and tolerable adoptive cellular immunotherapy.

[0160] In certain embodiments, the genome-edited immune effector cells contemplated herein are made more resistant to exhaustion by eliminating, reducing, or attenuating CBLB expression, CBLB activity, and / or CBLB-mediated signaling.

[0161] In various embodiments, contemplated genome editing compositions and methods include nuclease variants designed to bind to and cleave target sites in the Casitas B lineage (Cbl) lymphoma proto-oncogene B (CBLB) gene. In certain embodiments, contemplated nuclease variants can be used to introduce double-strand breaks in target polynucleotide sequences, which can be repaired by non-homologous end joining (NHEJ) in the absence of a polynucleotide template, e.g., a donor repair template, or by homology-directed repair (HDR), i.e., homologous recombination, in the presence of a donor repair template. In certain embodiments, contemplated nuclease variants can also be designed as nickases, which generate single-strand DNA breaks that can be repaired using the cellular base excision repair (BER) mechanism or homologous recombination in the presence of a donor repair template. NHEJ is an error-prone process that frequently results in the formation of small insertions and deletions that disrupt gene function. Homologous recombination requires homologous DNA as a template for repair and can be used to create an infinite variety of modifications specified by the introduction of donor DNA containing the desired sequence at the target site, flanked on both sides by sequences that retain homology to regions flanking the target site.

[0162] In one preferred embodiment, the genome editing composition contemplated herein comprises a homing endonuclease variant or megaTAL that targets the human CBLB gene.

[0163] In one preferred embodiment, the genome editing composition contemplated herein comprises a homing endonuclease variant or megaTAL and an endo-processing enzyme, such as Trex2.

[0164] In various embodiments, genome-edited cells are contemplated. Genome-edited cells comprise an edited CBLB gene, and the editing strategy is designed to reduce or eliminate CBLB expression. In certain embodiments, CAR T cells, genetically engineered TCR T cells, or DARIC T cells comprise an edited CBLB gene.

[0165] In various embodiments, DNA breaks are generated at the target site of the CBLB gene in T cells, e.g., immune effector cells, and NHEJ at the end of the cleaved genomic sequence can result in little or no CBLB expression in the cell, preferably resulting in T cells that lack or substantially lack functional CBLB expression and / or signaling, e.g., lack the ability to promote T cell exhaustion. Without wishing to be bound by any particular theory, T cells that lack functional CBLB expression are more resistant to immune suppression and T cell exhaustion and are therefore more durable and therapeutically effective.

[0166] In various other embodiments, a donor template is provided for repair of a broken CBLB genomic sequence. The CBLB gene is repaired with the template sequence by homologous recombination at the DNA break site. In certain embodiments, the repair template comprises a polynucleotide sequence that disrupts, and preferably substantially reduces or eliminates, functional CBLB expression.

[0167] In certain embodiments, the CBLB gene is repaired with a polynucleotide encoding an immunopotency enhancer, an immunosuppressive signal damper, or an engineered antigen receptor.

[0168] In certain embodiments, the CBLB gene is repaired with a polynucleotide encoding an immune enhancer, an immune suppressive signal damper, or an engineered antigen receptor, which is introduced into the CBLB gene so as to employ the endogenous CBLB promoter to transcriptionally control expression of the immune enhancer, the immune suppressive signal damper, or the engineered antigen receptor.

[0169] In a preferred embodiment, the genome editing compositions and methods contemplated herein are used to edit the human CBLB gene.

[0170] Thus, the methods and compositions contemplated herein represent a dramatic improvement over existing adoptive cell therapies.

[0171] Recombinant (i.e., genetically engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification, and related techniques and procedures may generally be performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology, as described and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning:A Practical Approach,vol.I&II(IRL Press,Oxford Univ.Press USA,1985);Current Protocols in Immunology(Edited by:John E.Coligan,Ada M.Kruisbeek,David H.Margulies,Ethan M.Shevach,Warren Strober 2001 John Wiley&Sons,NY,NY);Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,Kirstin Edwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK;Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991);Oligonucleotide Synthesis(N.Gait,Ed.,1984);Nucleic Acid The Hybridization(B.Hames&S.Higgins,Eds.,1985);Transcription and Translation(B.Hames&S.Higgins,Eds.,1984);Animal Cell Culture(R.Freshney,Ed.,1986);Perbal,A Practical Guide to Molecular Cloning(1984);Next-Generation Genome Sequencing(Janitz,2008 Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3rd Edition,2010 Humana Press);Immobilized Cells And Enzymes(IRL Press,1986);the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.);Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds.,1987,Cold Spring Harbor Laboratory);Harlow and Lane,Antibodies,(Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1998);Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (DMWeir and CC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and research articles such as Advances in Immunology.

[0172] B. Definition Before setting forth this disclosure in more detail, it may be helpful to its understanding to provide definitions of certain terms.

[0173] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of specific embodiments, preferred embodiments of the compositions, methods, and materials are described herein. For the purposes of this disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure.

[0174] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one or to more than one) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0175] The use of an alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0176] The term "and / or" should be understood to mean either one or both of its alternatives.

[0177] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that differs by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0178] In one embodiment, ranges, eg, 1 to 5, about 1 to 5, or about 1 to about 5, refer to each of the numbers encompassed by the range. For example, in one non-limiting and merely exemplary embodiment, the range "1 to 5" is equivalent to the expression 1, 2, 3, 4, 5, or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0, or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0179] As used herein, the term "substantially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces approximately the same effect, e.g., a physiological effect, as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0180] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" shall be understood to imply the inclusion of the stated step or element or group of steps or elements, but the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and that no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that no other elements are present that materially affect the activity or function of the recited elements.

[0181] Throughout this specification, reference to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the appearances of these phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the affirmative recitation of a feature in an embodiment serves as a basis for the exclusion of that feature in specific embodiments.

[0182] The term "ex vivo" generally refers to activities occurring outside of an organism, such as experiments or measurements performed in or on living tissue, preferably in an artificial environment outside of the organism that minimally alters natural conditions. In certain embodiments, "ex vivo" procedures involve living cells or tissues taken from an organism and cultured or conditioned in a laboratory setting, usually under sterile conditions, for a period typically of several hours or up to about 24 hours, but including up to 48 or 72 hours, depending on the circumstances. In certain embodiments, such tissues or cells can be collected and frozen, and later thawed for ex vivo processing. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissues are typically considered "in vitro," although in certain embodiments, the term can be used interchangeably with ex vivo.

[0183] The term "in vivo" generally refers to an activity that occurs inside an organism. In one embodiment, a cell's genome is genetically engineered, edited, or modified in vivo.

[0184] "Enhance" or "promote" or "increase" or "augment" or "potentiate" generally refers to the ability of a nuclease variant, genome editing composition, or genome-edited cell contemplated herein to produce, elicit, or cause a greater response (i.e., a physiological response) compared to the response caused by either a vehicle or control. Measurable responses can include increased catalytic activity, binding affinity, binding site specificity, binding site selectivity, persistence, cytolytic activity, and / or increased pro-inflammatory cytokines, among others that are apparent from the understanding in the art and described herein. An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more fold (e.g., 500, 1000 fold) (including all integers and decimal points therebetween and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response produced by the vehicle or control.

[0185] "Decrease" or "lower" or "reducing" or "reducing" or "weakening" or "destroying" or "inhibiting" or "attenuating" generally refers to the ability of a nuclease variant, genome editing composition, or genome-edited cell contemplated herein to produce, elicit, or cause a lower response (i.e., a physiological response) compared to the response caused by either a vehicle or control. Measurable responses can include decreases in off-target binding affinity, off-target cleavage specificity, T-cell exhaustion, and the like. The amount of "decreased" or "reduced" is typically a "statistically significant" amount and can include a reduction that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more fold (e.g., 500, 1000 fold) (including all integers and decimal points therebetween and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by the vehicle or control (reference response).

[0186] "Maintain," or "preserve," or "maintain," or "no change," or "no substantial change," or "no substantial decrease" generally refer to the ability of a nuclease variant, genome editing composition, or genome-edited cell contemplated herein to produce, elicit, or cause a substantially similar or equivalent physiological response (i.e., downstream effect) compared to the response caused by either a vehicle or control. An equivalent response is one that is significantly different or not measurably different from the reference response.

[0187] As used herein, the terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targets" describe the binding of one molecule to another molecule, e.g., a DNA-binding domain of a polypeptide binding to DNA, with a higher binding affinity than background binding. A binding domain is one in which it binds to a target molecule, e.g., a target molecule, with a binding affinity of about 10 5 M -1 or greater affinity or K a (i.e., the equilibrium association constant of a particular binding interaction in units of 1 / M). 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 More than K a A "high affinity" binding domain binds to a target site with at least 10 7 M -1 , at least 10 8 M -1 , at least 109 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 , or K exceeding it a refers to those binding domains having the

[0188] Alternatively, affinity may, in certain embodiments, be measured by measuring the specific binding interaction in units of M (e.g., 10 -5 M~10 -13 M or less) d The affinity of a nuclease variant comprising one or more DNA-binding domains for a DNA target site contemplated in certain embodiments can be readily determined using conventional techniques, such as yeast cell surface display, or by binding association or displacement assays using labeled ligands.

[0189] In one embodiment, the affinity of specific binding is about 2-fold greater than background binding, about 5-fold greater than background binding, about 10-fold greater than background binding, about 20-fold greater than background binding, about 50-fold greater than background binding, about 100-fold greater than background binding, or about 1000-fold or greater than background binding.

[0190] The terms "selectively bind" or "selectively bound" or "selectively binding" or "selectively target" describe the preferential binding of one molecule to a target molecule (target binding) in the presence of multiple off-target molecules. In certain embodiments, the HE or megaTAL selectively binds to the target DNA binding site about 5, 10, 15, 20, 25, 50, 100, or 1000 times more frequently than the HE or megaTAL binds to the off-target DNA target binding site.

[0191] "On-target" refers to the target site sequence.

[0192] "Off-target" refers to a sequence that is similar, but not identical to, the target site sequence.

[0193] A "target site" or "target sequence" is a chromosomal or extrachromosomal nucleic acid sequence that defines a portion of nucleic acid to which a binding molecule binds and / or cleaves, provided that conditions sufficient for binding and / or cleavage are present. When referring to a polynucleotide sequence or SEQ ID NO: that refers to only one strand of a target site or target sequence, it will be understood that the target site or target sequence bound and / or cleaved by a nuclease variant is double-stranded and includes the reference sequence and its complementary sequence. In a preferred embodiment, the target site is a sequence within the human CBLB gene.

[0194] "Recombination" refers to the process of exchanging genetic information between two polynucleotides, including, but not limited to, non-homologous end joining (NHEJ) and donor capture via homologous recombination. In this disclosure, "homologous recombination (HR)" refers to a specialized form of such exchange that occurs, for example, during repair of double-strand breaks in cells via the homology-directed repair (HDR) mechanism. This process is known variously as "non-crossover gene conversion" or "short-tract gene conversion" because it requires nucleotide sequence homology and uses a "donor" molecule as a template to repair a "target" molecule (i.e., one that has experienced a double-strand break), leading to the transmission of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transmission may involve mismatch correction of heteroduplex DNA between the damaged target and donor, and / or "synthesis-dependent single-strand annealing," which uses the donor to resynthesize the genetic information that will become part of the target, and / or related processes. Such specialized HR often results in alteration of the sequence of the target molecule such that some or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

[0195] "NHEJ" or "non-homologous end joining" refers to the repair of double-stranded breaks in the absence of a donor repair template or homologous sequence. NHEJ can result in insertions and deletions at the break site. NHEJ is mediated by several subpathways, each of which has different mutational consequences. The typical NHEJ pathway (cNHEJ) requires the KU / DNA-PKcs / Lig4 / XRCC4 complex to ligate and restore the ends with minimal processing, often resulting in precise repair of the break. Alternative NHEJ pathways (altNHEJ) are also active in repairing dsDNA breaks, but these pathways are significantly more mutagenic and often result in imprecise repair of breaks characterized by insertions and deletions. Without wishing to be bound by any particular theory, it is contemplated that modification of dsDNA breaks by end-processing enzymes such as exonucleases, e.g., Trex2, can increase the likelihood of imprecise repair.

[0196] "Cleavage" refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand cleavage are possible. Double-strand cleavage can occur as a result of two different single-strand cleavage events. DNA cleavage can result in the production of either blunt or staggered ends. In certain embodiments, the polypeptides and nuclease variants contemplated herein, such as homing endonuclease variants, megaTALs, etc., are used for targeted double-stranded DNA cleavage. The endonuclease cleavage recognition site can be on either DNA strand.

[0197] An "exogenous" molecule is a molecule that is not normally present in cells but is introduced into cells by one or more genetic, biochemical, or other methods. Representative exogenous molecules include, but are not limited to, small organic molecules, proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above molecules, or any complex containing one or more of the above molecules. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, biopolymer nanoparticles, calcium phosphate coprecipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer.

[0198] An "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. Additional endogenous molecules can include proteins.

[0199] "Gene" refers to a DNA region that encodes a gene product, as well as all DNA regions that regulate the production of the gene product, regardless of whether regulatory sequences flank the coding and / or transcribed sequence. Genes include, but are not limited to, promoter sequences, enhancers, silencers, insulators, boundary elements, terminators, polyadenylation sequences, post-transcriptional response elements, translational regulatory sequences, such as ribosome binding sites and internal ribosome entry sites, replication origins, substrate binding sites, and locus control regions.

[0200] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0201] As used herein, the terms "genetically engineered" or "genetically modified" refer to the chromosomal or extrachromosomal addition of foreign genetic material in the form of DNA or RNA to the total genetic material in a cell. The genetic modification may or may not be targeted to a specific site within the genome of the cell. In one embodiment, the genetic modification is site-specific. In one embodiment, the genetic modification is not site-specific.

[0202] As used herein, the term "genome editing" refers to the replacement, deletion, and / or introduction of genetic material at a target site within a cell's genome to restore, correct, disrupt, and / or modify the expression of a gene or gene product. In certain embodiments, contemplated genome editing involves introducing one or more nuclease variants into a cell, optionally in the presence of a donor repair template, to generate DNA damage at or adjacent to the target site within the cell's genome.

[0203] As used herein, the term "gene therapy" refers to the introduction of extra genetic material into the total genetic material in a cell for the purpose of restoring, correcting, or modifying the expression of a gene or gene product, or expressing a therapeutic polypeptide. In certain embodiments, the introduction of genetic material into the genome of a cell by genome editing for the purpose of restoring, correcting, disrupting, or modifying the expression of a gene or gene product, or expressing a therapeutic polypeptide, is considered gene therapy.

[0204] "Immune disorder" refers to a disease that evokes a response from the immune system. In certain embodiments, the term "immune disorder" refers to cancer, graft-versus-host disease, autoimmune disease, or immunodeficiency. In one embodiment, an immune disorder includes an infectious disease.

[0205] As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide without restriction and can invade nearby tissues.

[0206] As used herein, the term "malignant" refers to a cancer in which a group of tumor cells exhibit one or more of the following: uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., invasion of and destruction of adjacent tissue), and metastasis (i.e., spread to other locations in the body via the lymphatics or blood).

[0207] As used herein, the term "metastasize" refers to the spread of cancer from one part of the body to another. Tumors formed by spread cells are called "metastatic tumors" or "metastases." Metastatic tumors contain cells that are similar to the primary (primary) tumor.

[0208] As used herein, the terms "benign" or "non-malignant" refer to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limiting and typically do not invade or metastasize.

[0209] "Cancer cell" or "tumor cell" refers to an individual cell of a cancerous growth or tissue. A tumor generally refers to a swelling or lesion formed by the abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancers form tumors, but some, such as leukemia, do not necessarily form tumors. With respect to cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of tumors.

[0210] "Graft-versus-host disease" or "GVHD" refers to a complication that can occur after cell, tissue, or solid organ transplantation. GVHD can occur after stem cell or bone marrow transplantation, in which transplanted donor cells attack the recipient's body. Acute GVHD in humans occurs within approximately 60 days after transplantation and results in damage to the skin, liver, and internal organs due to the action of cytolytic lymphocytes. Chronic GVHD occurs later and is a systemic autoimmune disease that primarily affects the skin, resulting in polyclonal activation of B cells and excessive production of immunoglobulins and autoantibodies. Solid organ graft-versus-host disease (SOT-GVHD) occurs in two forms. The more common type is antibody-mediated, in which antibodies from a blood type O donor attack the recipient's red blood cells in a blood type A, B, or AB recipient, resulting in mild and transient hemolytic anemia. The second form of SOT-GVHD is a cellular type associated with high mortality, in which donor-derived T cells mount an immunological attack against immunologically foreign host tissues, most often in the skin, liver, gastrointestinal tract, and bone marrow, leading to complications in these organs.

[0211] "Graft-versus-leukemia" or "GVL" refers to an immune response against a person's leukemia cells by immune cells present in the donor's transplanted tissue, such as bone marrow or peripheral blood.

[0212] "Autoimmune disease" refers to a disease in which the body produces an immunogenic (i.e., immune system) response against some component of its own tissues. In other words, the immune system loses its ability to recognize some tissue or system in the body as "self" and targets and attacks it as if it were foreign. Illustrative examples of autoimmune diseases include, but are not limited to, arthritis, inflammatory bowel disease, Hashimoto's thyroiditis, Graves' disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like.

[0213] "Immunodeficiency" refers to a condition in which a patient's immune system is impaired by disease or the administration of chemicals. In this condition, the system becomes deficient in the number and types of blood cells needed to defend against foreign substances. Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immunodeficiency syndrome), SCID (severe combined immunodeficiency disease), selective IgA deficiency, common variable immunodeficiency, X-linked agammaglobulinemia, chronic granulomatous disease, hyper-IgM syndrome, Wiskott-Aldrich syndrome (WAS), and diabetes.

[0214] "Infectious disease" refers to a disease that can be transmitted from person to person or organism to organism and is caused by a microbial or viral agent (e.g., the common cold). Infectious diseases are known in the art and include, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia, gonorrhea), tuberculosis, HIV / AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.

[0215] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal exhibiting symptoms of an immune disorder that can be treated using nuclease variants, genome editing compositions, gene therapy vectors, genome editing vectors, genome-edited cells, and methods contemplated elsewhere herein. Suitable subjects (e.g., patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), livestock, and domestic animals or pets (such as cats or dogs). Non-human primates and preferably human subjects are included. Typical subjects include human patients who have, have been diagnosed with, or are at risk of having an immune disorder.

[0216] As used herein, the term "patient" refers to a subject who has been diagnosed with an immune disorder that can be treated using the nuclease variants, genome editing compositions, gene therapy vectors, genome editing vectors, genome edited cells, and methods contemplated elsewhere herein.

[0217] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated, such as cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. Treatment may optionally involve slowing the progression of the disease or condition. "Treatment" does not necessarily refer to the complete eradication or cure of the disease or condition or its associated symptoms.

[0218] As used herein, "prevent" and similar terms such as "prevention," "prevented," "preventing," and the like refer to an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition, such as cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0219] As used herein, the phrase "ameliorating at least one symptom of" refers to reducing one or more symptoms of the disease or condition that the subject is being treated for, such as cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency.In certain embodiments, the disease or condition being treated is cancer, and the one or more symptoms that are alleviated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, swollen lymph nodes, distended or painful abdomen (due to enlarged abdominal organs), bone or joint pain, fractures, unintentional weight loss, loss of appetite, night sweats, persistent low-grade fever, and reduced urination (due to renal insufficiency).

[0220] As used herein, the term "amount" refers to an "effective amount" or "effective quantity" of a nuclease variant, genome editing composition, or genome edited cell sufficient to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0221] A "prophylactically effective amount" refers to an amount of a nuclease variant, genome editing composition, or genome-edited cell sufficient to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactically effective amount is less than a therapeutically effective amount, since a prophylactic dose is used in a subject before or at an earlier stage of disease.

[0222] A "therapeutically effective amount" of a nuclease variant, genome editing composition, or genome-edited cell may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or adverse effects are outweighed by therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of a contemplated composition to be administered in a particular embodiment can be determined by a physician in light of the specifications and taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and pathological condition.

[0223] C. Nuclease Variants Nuclease variants contemplated in certain embodiments herein are suitable for genome editing target sites within CBLB genes and comprise one or more DNA-binding domains, one or more DNA-cleavage domains (e.g., one or more endonuclease and / or exonuclease domains), and, optionally, one or more linkers as contemplated herein. The terms "reprogrammed nuclease," "engineered nuclease," or "nuclease variant" are used interchangeably and refer to a nuclease that comprises one or more DNA-binding domains and one or more DNA-cleavage domains, where the nuclease has been designed and / or modified from a parent or naturally occurring nuclease to bind to and cleave a double-stranded DNA target sequence within a CBLB gene.

[0224] In certain embodiments, the nuclease variant binds to and cleaves a target sequence in exon 6 of the CBLB gene, preferably at SEQ ID NO: 20 in exon 6 of the CBLB gene, and more preferably at the sequence "ATTC" within SEQ ID NO: 20 in exon 6 of the CBLB gene.

[0225] Nuclease variants can be designed and / or modified from naturally occurring nucleases or from previous nuclease variants. In certain embodiments, contemplated nuclease variants can further comprise one or more additional functional domains, such as a 5'-3' exonuclease, a 5-3' alkaline exonuclease, a 3'-5' exonuclease (e.g., Trex2), a 5' flap endonuclease, a helicase, a template-dependent DNA polymerase, or an endo-processing enzyme domain of an endo-processing enzyme that exhibits template-independent DNA polymerase activity.

[0226] Illustrative examples of nuclease variants that bind to and cleave target sequences within CBLB genes include, but are not limited to, homing endonuclease (meganuclease) variants and megaTALs.

[0227] 1. Homing endonuclease (meganuclease) variants In various embodiments, the homing endonuclease or meganuclease is reprogrammed to introduce a double-stranded break (DSB) at a target site within the CBLB gene. In certain embodiments, the homing endonuclease variant introduces a double-stranded break in exon 6 of the CBLB gene, preferably at SEQ ID NO: 20 in exon 6 of the CBLB gene, and more preferably at the sequence "ATTC" within SEQ ID NO: 20 in exon 6 of the CBLB gene.

[0228] The terms "homing endonucleases" and "meganucleases" are used interchangeably to refer to naturally occurring homing endonucleases that recognize 12-45 base pair cleavage sites and are generally classified into five families based on sequence and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK.

[0229] A "reference homing endonuclease" or "reference meganuclease" refers to a wild-type homing endonuclease or homing endonuclease found in nature. In one embodiment, a "reference homing endonuclease" refers to a wild-type homing endonuclease that has been modified to increase its basal activity.

[0230] "Engineered homing endonuclease," "reprogrammed homing endonuclease," "homing endonuclease variant," "engineered meganuclease," "reprogrammed meganuclease," or "meganuclease variant" refers to a homing endonuclease that contains one or more DNA-binding domains and one or more DNA-cleavage domains, and the homing endonuclease has been designed and / or modified from a parent or naturally occurring homing endonuclease to bind to and cleave a DNA target sequence within a CBLB gene. The homing endonuclease variant may be designed and / or modified from a naturally occurring homing endonuclease or another homing endonuclease variant. In certain embodiments, contemplated homing endonuclease variants may further comprise one or more additional functional domains, e.g., an endo-processing enzyme domain of a 5'-3' exonuclease, a 5-3' alkaline exonuclease, a 3'-5' exonuclease (e.g., Trex2), a 5' flap endonuclease, a helicase, a template-dependent DNA polymerase, or an endo-processing enzyme that exhibits template-independent DNA polymerase activity.

[0231] Homing endonuclease (HE) variants do not exist in nature and can be obtained by recombinant DNA technology or random mutagenesis.HE variants can be obtained by making one or more amino acid modifications in naturally occurring HE or HE variants, for example, by mutating, substituting, adding, or deleting one or more amino acids.In certain embodiments, HE variants comprise one or more amino acid modifications in DNA recognition interface.

[0232] In certain embodiments, contemplated HE variants may further comprise one or more linkers and / or additional functional domains, e.g., an endo-processing enzyme domain of an endo-processing enzyme exhibiting 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerase, or template-independent DNA polymerase activity. In certain embodiments, the HE variant is introduced into T cells along with an endo-processing enzyme exhibiting 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerase, or template-independent DNA polymerase activity. The HE variant and the 3' processing enzyme can be introduced separately, for example, in different vectors or separate mRNAs, or together, for example, as a fusion protein or in a polycistronic construct separated by a viral self-cleaving peptide or IRES element.

[0233] The "DNA recognition interface" refers to the HE amino acid residues that interact with the nucleic acid target base, as well as adjacent residues. For each HE, the DNA recognition interface contains an extensive network of side chain-side chain and side chain-DNA contact points, most of which are necessarily unique for recognizing a specific nucleic acid target sequence. Therefore, the amino acid sequence of the DNA recognition interface corresponding to a specific nucleic acid sequence will vary significantly and be characteristic of any natural or HE variant. As a non-limiting example, HE variants contemplated in certain embodiments can be generated by constructing a library of HE variants that vary in one or more amino acid residues located in the DNA recognition interface of a natural HE (or a previously generated HE variant). The library can be screened for target cleavage activity against each predicted CBLB target site using a cleavage assay (see, for example, Jarjour et al., 2009. Nuc. Acids Res. 37(20):6871-6880).

[0234] LAGLIDADG homing endonucleases (LHEs) are the most thoroughly studied family of homing endonucleases. They are encoded primarily in archaea and in the organelle DNA of green algae and fungi, and exhibit the highest overall DNA recognition specificity. LHEs contain one or two LAGLIDADG catalytic motifs per protein chain, functioning as homodimers or single-chain monomers, respectively. Structural studies of LAGLIDADG proteins have identified a highly conserved core structure characterized by an αββαββα fold (Stoddard 2005), of which the LAGLIDADG motif resides in the first helix. The highly efficient and specific cleavage by LHEs represents a protein scaffold for generating novel, highly specific endonucleases. However, engineering an LHE to bind to and cleave non-natural or non-canonical target sites requires selection of an appropriate LHE scaffold, examination of the target locus, selection of a putative target site, and extensive modification of the LHE to alter its DNA contact points and cleavage specificity at up to two-thirds of the base pair positions in the target site.

[0235] In one embodiment, LHEs from which reprogrammed LHEs or LHE variants can be engineered include, but are not limited to, I-CreI and I-SceI.

[0236] Examples of LHEs from which reprogrammed LHEs or LHE variants can be engineered include I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, and I-GzeMIII. , I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, and I-Vdi141I.

[0237] In one embodiment, the reprogrammed LHE or LHE variant is selected from the group consisting of an I-CpaMI variant, an I-HjeMI variant, an I-OnuI variant, an I-PanMI variant, and an I-SmaMI variant.

[0238] In one embodiment, the reprogrammed LHE or LHE variant is an I-OnuI variant. See, e.g., SEQ ID NOs: 6-12.

[0239] In one embodiment, a reprogrammed I-OnuI targeting the CBLB gene The LHE or I-OnuI variant was generated from the native I-OnuI or a biologically active fragment thereof (SEQ ID NOs: 1-5). In a preferred embodiment, the reprogrammed I-OnuI LHE or I-OnuI variant targeting the human CBLB gene was generated from an existing I-OnuI variant. In one embodiment, the reprogrammed I-OnuI LHE was generated against the human CBLB gene target site shown in SEQ ID NO: 20.

[0240] In certain embodiments, the reprogrammed I-OnuI LHE or I-OnuI variant that binds to and cleaves the human CBLB gene comprises one or more amino acid substitutions in the DNA recognition interface. In certain embodiments, the I-OnuI LHE that binds to and cleaves the CBLB gene is I-OnuI (Taekuchi 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity with the DNA recognition interface of an I-OnuI LHE variant as set forth in any one of SEQ ID NOs: 6-12, a biologically active fragment thereof, and / or a further variant thereof.

[0241] In one embodiment, the I-OnuI LHE that binds to and cleaves the human CBLB gene comprises at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 99% sequence identity with the DNA recognition interface of I-OnuI (Taekuchi et al. 2011. Proc Natl Acad Sci USA 2011 Aug 9;108(32):13077-13082) or an I-OnuI LHE variant set forth in SEQ ID NOs: 6-12, a biologically active fragment thereof, and / or a further variant thereof.

[0242] In a specific embodiment, I-OnuI binds to and cleaves the human CBLB gene. The LHE variant comprises one or more amino acid substitutions or modifications in the DNA recognition interface of I-OnuI set forth in any one of SEQ ID NOs: 1-12, a biologically active fragment thereof, and / or a further variant thereof.

[0243] In a specific embodiment, I-OnuI binds to and cleaves the human CBLB gene. The LHE variant comprises one or more amino acid substitutions or modifications in the DNA recognition interface of I-OnuI (SEQ ID NOs: 1-5) or an I-OnuI variant set forth in any one of SEQ ID NOs: 6-12, a biologically active fragment thereof, and / or a further variant thereof, in particular in the subdomains located at positions 24-50, 68-82, 180-203, and 223-240.

[0244] In certain embodiments, the HE variant comprises one or more amino acid substitutions in the DNA recognition interface at amino acid positions selected from the group consisting of 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76, 78, 80, 82, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of I-OnuI (SEQ ID NOs: 1-5) or an I-OnuI variant set forth in any one of SEQ ID NOs: 6-12, a biologically active fragment thereof, and / or a variant thereof.

[0245] In certain embodiments, the HE variants have one or more amino acid substitutions in a sequence similar to or similar to 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 142, 144, 146, 148, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, At amino acid positions selected from the group consisting of 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240.

[0246] In a specific embodiment, I-OnuI binds to and cleaves the human CBLB gene. The LHE variants comprise 5, 10, 15, 20, 25, 30, 35, or 40 or more amino acid substitutions or modifications in the DNA recognition interface of I-OnuI (SEQ ID NOs: 1-5) or an I-OnuI variant set forth in any one of SEQ ID NOs: 6-12, a biologically active fragment thereof, and / or a further variant thereof, particularly in the subdomains located at positions 24-50, 68-82, 180-203, and 223-240.

[0247] In a specific embodiment, I-OnuI binds to and cleaves the human CBLB gene. LHE variants may have 5, 10, 15, 20, 25, 30, 35, or 40 or more amino acid substitutions or modifications in I-OnuI (SEQ ID NOS: 1-5) or an I-OnuI variant set forth in any one of SEQ ID NOS: 6-12, a biologically active fragment thereof, and / or a variant thereof, such as 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 1109, 1111, 1120, 1121, 1130, 1140, 1150, 1160, 1170, 1180, 1190, At an amino acid position selected from the group consisting of 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76, 78, 80, 82, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240.

[0248] In a specific embodiment, I-OnuI binds to and cleaves the human CBLB gene. LHE variants may have 5, 10, 15, 20, 25, 30, 35, or 40 or more amino acid substitutions or modifications in any one of I-OnuI (SEQ ID NOS: 1-5) or I-OnuI variants set forth in any one of SEQ ID NOS: 6-12, biologically active fragments thereof, and / or variants thereof, at positions 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76, 78, 80, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 142, 144, 146, 148, 150, 151, 152, At amino acid positions selected from the group consisting of 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240.

[0249] In one embodiment, an I-OnuI LHE variant that binds to and cleaves the human CBLB gene contains one or more amino acid substitutions or modifications at additional positions located anywhere throughout the I-OnuI sequence. Residues that may be substituted and / or modified include, but are not limited to, amino acids that contact the nucleic acid target or interact with the nucleic acid backbone or nucleotide bases, either directly or via water molecules. In one non-limiting example, an I-OnuI LHE variant contemplated herein that binds to and cleaves the human CBLB gene contains one or more substitutions and / or modifications, preferably at least 5, preferably at least 10, preferably at least 15, preferably at least 20, more preferably at least 25, more preferably at least 30, even more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions, in I-OnuI (SEQ ID NOS: 1-5) or an I-OnuI variant set forth in any one of SEQ ID NOS: 6-12, its biological activity, and / or variants thereof at at least one position selected from the group consisting of: 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 78, 80, 92, 116, 138, 143, 159, 168, 178, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 207, 223, 225, 227, 232, 236, and 238.

[0250] In certain embodiments, the HE variant cleaves the CBLB exon 6 target site and is selected from the group consisting of I-OnuI (SEQ ID NOs: 1-5) and I-OnuI variants set forth in any one of SEQ ID NOs: 6-12, biologically active fragments thereof, and / or further variants thereof, including the following amino acid substitutions: S24C, L26R, L26G, R28D, R28Y, R30H, N32A, N32S, K34D, K34V, S35L, S36R, V37A, V37S, S40R, E42R, G44A, G44S, Q46E, T48V, T48S, V68T, V68K, A70Y, S72A, S78R, K80Q, D92G, V116L , L138M, T143N, S159P, F168L, E178D, C180S, F182V, F182M, N184E, I186K, I186M, S188R, S188N, K189R, S190N, K191P, K191N, L192V, G193K, G193I, Q195G, Q195R, Q197R, V199R, S201G, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0251] In some embodiments, the HE variant cleaves a CBLB target site and is selected from the group consisting of I-OnuI (SEQ ID NOs: 1-5) and I-OnuI variants set forth in any one of SEQ ID NOs: 6-12, biologically active fragments thereof, and / or further variants thereof, including the following amino acid substitutions: S24C, L26R, R28D, N32A, K34D, S35L, S36R, V37A, S40R, E42R, G44A, Q46E, T48V, V68T, A70Y, S72A, S78R, K80Q , L138M, T143N, F168L, E178D, C180S, F182V, N184E, I186K, S188R, K189R, K191P, L192V, G193K, Q195G, Q197R, V199R, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0252] In certain embodiments, the HE variant cleaves the CBLB target site and is selected from the group consisting of I-OnuI (SEQ ID NOs: 1-5) and I-OnuI variants set forth in any one of SEQ ID NOs: 6-12, biologically active fragments thereof, and / or further variants thereof, including the following amino acid substitutions: S24C, L26R, R28D, N32A, K34D, S35L, S36R, V37A, S40R, E42R, G44A, Q46E, T48V, V68T, A70Y, S72A, S78R, K80Q, L138M, At least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following: T143N, S159P, F168L, E178D, C180S, F182M, N184E, I186M, S188N, S190N, K191N, L192V, G193I, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0253] In certain embodiments, the HE variant cleaves the CBLB target site and is identical to I-OnuI (SEQ ID NOS: 1-5) or an I-OnuI variant set forth in any one of SEQ ID NOS: 6-12, a biologically active fragment thereof, and / or a further variant thereof, with the following amino acid substitutions: S24C, L26R, R28D, N32A, K34D, S35L, S36R, V37A, S40R, E42R, G44S, Q46E, T48S, V68T, A70Y, S72A, S78R, K80Q, D92G, V116L, L and at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of 138M, T143N, S159P, F168L, E178D, C180S, F182M, N184E, I186M, S188N, S190N, K191N, L192V, G193I, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0254] In certain embodiments, the HE variant cleaves the CBLB target site and is selected from the group consisting of I-OnuI (SEQ ID NOS: 1-5) and I-OnuI variants set forth in any one of SEQ ID NOS: 6-12, biologically active fragments thereof, and / or further variants thereof, including the following amino acid substitutions: S24C, L26R, R28D, R30H, N32A, K34V, S35L, S36R, V37S, S40R, E42R, G44S, Q46E, T48V, V68T, V68K, A70Y, S72A, S78R, K80Q, L and at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of 138M, T143N, S159P, F168L, E178D, C180S, F182M, N184E, I186M, S188N, S190N, K191N, L192V, G193I, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0255] In certain embodiments, the HE variant cleaves the CBLB target site and is selected from the group consisting of I-OnuI (SEQ ID NOS: 1-5) and I-OnuI variants set forth in any one of SEQ ID NOS: 6-12, biologically active fragments thereof, and / or further variants thereof, including the following amino acid substitutions: S24C, L26G, R28Y, R30H, N32S, K34V, S35L, S36R, V37S, S40R, E42R, G44S, Q46E, T48S, V68T, A70Y, S72A, S78R, K80Q, V116L, L and at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of 138M, T143N, S159P, F168L, E178D, C180S, F182M, N184E, I186M, S188N, S190N, K191N, L192V, G193I, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0256] In certain embodiments, the HE variant cleaves the CBLB target site and is selected from the group consisting of I-OnuI (SEQ ID NOS: 1-5) and I-OnuI variants set forth in any one of SEQ ID NOS: 6-12, biologically active fragments thereof, and / or further variants thereof, including the following amino acid substitutions: S24C, L26R, R28D, R30H, N32A, K34V, S35L, S36R, V37S, S40R, E42R, G44S, Q46E, T48V, V68T, A70Y, S72A, S78R, K80Q, V116L, L and at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of 138M, T143N, S159P, F168L, E178D, C180S, F182V, N184E, I186K, S188R, K189R, K191P, L192V, G193K, Q195G, Q197R, V199R, S201G, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0257] In certain embodiments, the HE variant cleaves the CBLB target site and is identical to I-OnuI (SEQ ID NOs: 1-5) or an I-OnuI variant set forth in any one of SEQ ID NOs: 6-12, a biologically active fragment thereof, and / or a further variant thereof, with the following amino acid substitutions: S24C, L26R, R28D, N32A, K34D, S35L, S36R, V37A, S40R, E42R, G44A, Q46E, T48V, V68T, A70Y, S72A, S78R, K80Q, D92G, L138 M, T143N, S159P, F168L, E178D, C180S, F182M, N184E, I186M, S188N, S190N, K191N, L192V, G193I, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, F232H, D236E, and V238I.

[0258] In a specific embodiment, I-OnuI binds to and cleaves the human CBLB gene. The LHE variant comprises an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 6-12, or a biologically active fragment thereof.

[0259] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 6-12, or a biologically active fragment thereof.

[0260] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.

[0261] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence set forth in SEQ ID NO: 7, or a biologically active fragment thereof.

[0262] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence set forth in SEQ ID NO: 8, or a biologically active fragment thereof.

[0263] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence set forth in SEQ ID NO: 9, or a biologically active fragment thereof.

[0264] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence set forth in SEQ ID NO: 10, or a biologically active fragment thereof.

[0265] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence set forth in SEQ ID NO: 11, or a biologically active fragment thereof.

[0266] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence set forth in SEQ ID NO: 12, or a biologically active fragment thereof.

[0267] 2.magaTAL In various embodiments, the megaTAL containing the homing endonuclease variant is reprogrammed to introduce a double-strand break (DSB) at a target site in the CBLB gene. In a specific embodiment, the megaTAL introduces a DSB in exon 6 of the CBLB gene, preferably at SEQ ID NO: 20 in exon 6 of the CBLB gene, and more preferably at the sequence "ATTC" within SEQ ID NO: 20 in exon 6 of the CBLB gene.

[0268] "megaTAL" refers to a polypeptide comprising a TALE DNA binding domain and a homing endonuclease variant that binds to and cleaves a DNA target sequence within a CBLB gene, and optionally includes one or more linkers and / or additional functional domains, such as a 5-3' exonuclease, a 5-3' alkaline exonuclease, a 3-5' exonuclease (e.g., Trex2), a 5' flap endonuclease, a helicase, or an endo-processing enzyme domain of an endo-processing enzyme that exhibits template-independent DNA polymerase activity.

[0269] In certain embodiments, a megaTAL can be introduced into a cell together with an end-processing enzyme that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerase, or template-independent DNA polymerase activity. The megaTAL and the 3' processing enzyme can be introduced separately, for example, on different vectors or separate mRNAs, or together, for example, as fusion proteins, or in a polycistronic construct separated by a viral self-cleaving peptide or IRES element.

[0270] A "TALE DNA-binding domain" is the DNA-binding portion of a transcription activator-like effector (TALE or TAL-effector) that mimics a plant transcription activator to manipulate plant transcripts (see, e.g., Kay et al., 2007. Science 318:648-651). In certain embodiments, contemplated TALE DNA-binding domains are engineered de novo or from naturally occurring TALEs, such as AvrBs3 from Xanthomonas campestris pv. vesicatoria, Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonas euvesicatoria, and Xanthomonas oryzae, and brg11 and hpx17 from Ralstonia solanacearum. Illustrative examples of TALE proteins for deriving and engineering DNA binding domains are disclosed in U.S. Patent No. 9,017,967 and the references cited therein, all of which are incorporated herein by reference in their entirety.

[0271] In certain embodiments, megaTALs comprise a TALE DNA-binding domain, which comprises one or more repeat units that are involved in the binding of the TALE DNA-binding domain to its corresponding target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length. Each TALE DNA-binding domain repeat unit typically contains one or two DNA-binding residues constituting a Repeat Variable Di-Residue (RVD) at positions 12 and / or 13 of the repeat. The natural (canonical) code for DNA recognition of these TALE DNA-binding domains has been determined such that the HD sequence at positions 12 and 13 results in binding to cytosine (C), NG binds to T, NI binds to A, NN binds to G or A, and NG binds to T. In certain embodiments, non-canonical (atypical) RVDs are contemplated.

[0272] Illustrative examples of non-canonical RVDs suitable for use in certain megaTALs contemplated in certain embodiments include, but are not limited to, HH, KH, NH, NK, NQ, RH, RN, SS, NN, SN, KN for the recognition of guanine (G); NI, KI, RI, HI, SI for the recognition of adenine (A); NG, HG, KG, RG for the recognition of thymine (T); RD, SD, HD, ND, KD, YG for the recognition of cytosine (C); NV, HN for the recognition of A or G; and H*, HA, KA, N*, NA, NC, NS, RA, S* (where * denotes the absence of an amino acid at position 13) for the recognition of A or T or G or C. Additional illustrative examples of RVDs suitable for use in certain megaTALs contemplated in certain embodiments further include those disclosed in U.S. Patent No. 8,614,092, which is incorporated herein by reference in its entirety.

[0273] In certain embodiments, megaTALs contemplated herein comprise a TALE DNA binding domain comprising 3-30 repeat units. In certain embodiments, megaTALs comprise 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, or 30 TALE DNA binding domain repeat units. In preferred embodiments, megaTALs contemplated herein comprise a TALE DNA binding domain comprising 5-15 repeat units, more preferably 7-15 repeat units, more preferably 9-15 repeat units, and more preferably 9, 10, 11, 12, 13, 14, or 15 repeat units.

[0274] In certain embodiments, megaTALs contemplated herein comprise a TALE DNA-binding domain comprising 3 to 30 repeat units and an additional single truncated TALE repeat unit comprising 20 amino acids located at the C-terminus of a set of TALE repeat units, i.e., an additional C-terminal half-TALE DNA-binding domain repeat unit (amino acids -20 to -1 of the C-cap disclosed elsewhere herein (see below)). Thus, in certain embodiments, megaTALs contemplated herein comprise a TALE DNA-binding domain comprising 3.5 to 30.5 repeat units. In certain embodiments, the megaTAL comprises 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, 15.5, 16.5, 17.5, 18.5, 19.5, 20.5, 21.5, 22.5, 23.5, 24.5, 25.5, 26.5, 27.5, 28.5, 29.5, or 30.5 TALE DNA binding domain repeat units. In a preferred embodiment, the megaTAL contemplated herein comprises a TALE DNA binding domain comprising 5.5 to 15.5 repeat units, more preferably 7.5 to 15.5 repeat units, more preferably 9.5 to 15.5 repeat units, and more preferably 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, or 15.5 repeat units.

[0275] In certain embodiments, a megaTAL comprises a TAL effector structure comprising an "N-terminal domain (NTD)" polypeptide, one or more TALE repeat domains / units, a "C-terminal domain (CTD)" polypeptide, and a homing endonuclease variant. In some embodiments, the NTD, TALE repeat, and / or CTD domains are derived from the same species. In other embodiments, one or more of the NTD, TALE repeat, and / or CTD domains are derived from different species.

[0276] As used herein, the term "N-terminal domain (NTD)" polypeptide refers to the sequence adjacent to the N-terminal portion or fragment of a naturally occurring TALE DNA-binding domain. The NTD sequence, if present, can be of any length, so long as the TALE DNA-binding domain repeat unit retains the ability to bind to DNA. In certain embodiments, the NTD polypeptide comprises at least 120 to at least 140 or more amino acids N-terminal to the TALE DNA-binding domain (where 0 is amino acid 1 of the most N-terminal repeat unit). In certain embodiments, the NTD polypeptide comprises at least about 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or at least 140 amino acids N-terminal to the TALE DNA-binding domain. In one embodiment, a megaTAL contemplated herein comprises an NTD polypeptide from at least about amino acids +1 to +122 to at least about +1 to +137 of a Xanthomonas TALE protein (where 0 is amino acid 1 of the most N-terminal repeat unit). In specific embodiments, the NTD polypeptide comprises at least about 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137 amino acids N-terminal to the TALE DNA-binding domain of a Xanthomonas TALE protein. In one embodiment, a megaTAL contemplated herein comprises an NTD polypeptide from at least amino acids +1 to +121 of a Ralstonia TALE protein (where 0 is amino acid 1 of the most N-terminal repeat unit). In certain embodiments, the NTD polypeptide comprises at least about 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137 amino acids N-terminal to the TALE DNA binding domain of a Ralstonia TALE protein.

[0277] As used herein, the term "C-terminal domain (CTD)" polypeptide refers to the sequence adjacent to the C-terminal portion or fragment of a naturally occurring TALE DNA-binding domain. The CTD sequence, if present, can be of any length, so long as the TALE DNA-binding domain repeat unit retains the ability to bind to DNA. In certain embodiments, the CTD polypeptide comprises at least 20 to at least 85 or more amino acids C-terminal to the last full repeat of the TALE DNA-binding domain (the first 20 amino acids are a half-repeat unit C-terminal to the last C-terminal full repeat unit). In certain embodiments, the CTD polypeptide comprises at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or at least 85 amino acids C-terminal to the last complete repeat of the TALE DNA binding domain. In one embodiment, a megaTAL contemplated herein comprises a CTD polypeptide of at least about amino acids -20 to -1 of a Xanthomonas TALE protein (-20 is amino acid 1 of the half-repeat unit C-terminal to the last C-terminal full repeat unit). In a specific embodiment, the CTD polypeptide comprises at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids C-terminal to the last full repeat of the TALE DNA-binding domain of a Xanthomonas TALE protein. In one embodiment, a megaTAL contemplated herein comprises a CTD polypeptide of at least about amino acids -20 to -1 of a Ralstonia TALE protein (-20 is amino acid 1 of the half-repeat unit C-terminal to the last C-terminal full repeat unit).In certain embodiments, the CTD polypeptide comprises at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid C-terminal to the last complete repeat of the TALE DNA binding domain of a Ralstonia TALE protein.

[0278] In certain embodiments, the megaTAL contemplated herein comprises a fusion polypeptide comprising a TALE DNA-binding domain engineered to bind to a target sequence, a homing endonuclease reprogrammed to bind to and cleave the target sequence, and, optionally, an NTD and / or CTD polypeptide, optionally linked to one or more linker polypeptides contemplated elsewhere herein. Without wishing to be bound by any particular theory, it is contemplated that the megaTAL comprising the TALE DNA-binding domain and, optionally, the NTD and / or CTD polypeptide is fused to a linker polypeptide that is further fused to a homing endonuclease variant. Thus, the TALE DNA-binding domain binds to a DNA target sequence that is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides of the target sequence bound by the DNA-binding domain of the homing endonuclease variant. In this way, the megaTAL contemplated herein increases the specificity and efficiency of genome editing.

[0279] In one embodiment, the megaTAL comprises a homing endonuclease variant and a TALE DNA binding domain that binds to a nucleotide sequence within about 4, 5, or 6 nucleotides, preferably 5 or 6 nucleotides upstream of the binding site of the reprogrammed homing endonuclease.

[0280] In one embodiment, the megaTAL comprises a homing endonuclease variant and a TALE DNA binding domain that binds to the nucleotide sequence shown in SEQ ID NO: 21, which is 5 nucleotides upstream of the nucleotide sequence (SEQ ID NO: 20) to which the homing endonuclease variant binds and cleaves (i.e., there are 4 nucleotides between the TALE binding site and the HE binding site). In a preferred embodiment, the megaTAL target sequence is SEQ ID NO: 22.

[0281] In certain embodiments, megaTALs contemplated herein comprise one or more TALE DNA-binding repeat units and one or more of I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI , I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, I-Vdi141I, and variants thereof, or preferably an LHE variant engineered or reprogrammed from an LHE selected from the group consisting of I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, SmaMI, and variants thereof, or more preferably I-OnuI and variants thereof.

[0282] In certain embodiments, megaTALs contemplated herein comprise an NTD, one or more TALE DNA-binding repeat units, a CTD, and one or more of: I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I- and an LHE variant selected from the group consisting of NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, I-Vdi141I, and variants thereof, or preferably I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, SmaMI, and variants thereof, or more preferably I-OnuI and variants thereof.

[0283] In certain embodiments, megaTALs contemplated herein comprise an NTD, about 9.5 to about 15.5 TALE DNA-binding repeat units, and one or more of: I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-Ncr and an LHE variant selected from the group consisting of I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, I-Vdi141I, and variants thereof, or preferably I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, SmaMI, and variants thereof, or more preferably I-OnuI and variants thereof.

[0284] In certain embodiments, a megaTAL contemplated herein comprises an NTD of about 122 amino acids to 137 amino acids, about 9.5, about 10.5, about 11.5, about 12.5, about 13.5, about 14.5, or about 15.5 binding repeat units, a CTD of about 20 amino acids to about 85 amino acids, and an I-OnuI LHE variant. In certain embodiments, any one, two, or all of the NTD, DNA-binding domain, and CTD can be designed from the same or different species in any suitable combination.

[0285] In certain embodiments, a megaTAL contemplated herein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13-19.

[0286] In certain embodiments, the megaTAL-Trex2 fusion protein contemplated herein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 13-19 and 38.

[0287] In certain embodiments, the megaTAL comprises a TALE DNA binding domain, and the I-OnuI LHE variant binds to and cleaves the nucleotide sequence set forth in SEQ ID NO:22.

[0288] 3. Endo-processing enzymes In certain embodiments, contemplated genome editing compositions and methods include editing a cell's genome using one or more copies of a nuclease variant and an end-processing enzyme. In certain embodiments, a single polynucleotide encodes a homing endonuclease variant and an end-processing enzyme, separated by a linker, a self-cleaving peptide sequence, such as a 2A sequence, or an IRES sequence. In certain embodiments, the genome editing composition comprises a polynucleotide encoding the nuclease variant and a separate polynucleotide encoding the end-processing enzyme. In certain embodiments, the genome editing composition comprises a polynucleotide encoding the homing endonuclease variant and an end-processing enzyme in a single fusion polypeptide. In one embodiment, the fusion polypeptide comprises a megaTAL and one or more copies of the end-processing enzyme, each separated by a self-cleaving peptide.

[0289] The term "endo-processing enzyme" refers to an enzyme that modifies exposed termini of a polynucleotide chain. Polynucleotides can be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA, double-stranded hybrids of DNA and RNA, and synthetic DNA (e.g., containing bases other than A, C, G, and T). Endo-processing enzymes can modify exposed polynucleotide chain termini by adding one or more nucleotides, removing one or more nucleotides, removing or modifying phosphate groups, and / or removing or modifying hydroxyl groups. Endo-processing enzymes can modify termini at endonuclease cleavage sites or termini generated by other chemical or mechanical means, such as shearing (e.g., by passing through a fine-gauge needle, heating, sonication, small-bead shaking, and spraying), ionizing radiation, ultraviolet irradiation, oxygen radicals, chemical hydrolysis, and chemotherapeutic agents.

[0290] In certain embodiments, genome editing compositions and methods contemplated in certain embodiments include editing a cell's genome using a homing endonuclease variant or megaTAL, and a DNA end-processing enzyme.

[0291] The term "DNA end-processing enzyme" refers to an enzyme that modifies exposed ends of DNA. DNA end-processing enzymes can modify blunt ends or staggered ends (ends with 5' or 3' overhangs). DNA end-processing enzymes can modify single-stranded or double-stranded DNA. DNA end-processing enzymes can modify ends at endonuclease cleavage sites or ends generated by other chemical or mechanical means, such as shearing (e.g., by passing through a fine-gauge needle, heating, sonication, small-bead shaking, and spraying), ionizing radiation, ultraviolet irradiation, oxygen radicals, chemical hydrolysis, and chemotherapeutic agents. DNA end-processing enzymes can modify exposed DNA ends by adding one or more nucleotides, removing one or more nucleotides, removing or modifying phosphate groups, and / or removing or modifying hydroxyl groups.

[0292] Illustrative examples of DNA end-processing enzymes suitable for use in certain embodiments contemplated herein include, but are not limited to, 5'-3' exonucleases, 5'-3' alkaline exonucleases, 3'-5' exonucleases, 5' flap endonucleases, helicases, phosphatases, hydrolases, and template-independent DNA polymerases.

[0293] Additional illustrative examples of DNA end-processing enzymes suitable for use in certain embodiments contemplated herein include Trex2, Trex1, Trex1 without a transmembrane domain, Apollo, Artemis, DNA2, Exo1, ExoT, ExoIII, Fen1, Fan1, MreII, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, lambda exonuclease, Sox, Vaccine These include, but are not limited to, senior DNA polymerase, exonuclease I, exonuclease III, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7-exonuclease gene 6, avian myeloblastosis virus integration protein (IN), Bloom, Antarctic phosphatase, alkaline phosphatase, polynucleotide kinase (PNK), ApeI, mung bean nuclease, Hex1, TTRAP (TDP2), Sgs1, Sae2, CUP, Pol mu, Pol lambda, MUS81, EME1, EME2, SLX1, SLX4, and UL-12.

[0294] In certain embodiments, genome editing compositions and methods for editing a cellular genome contemplated herein include a homing endonuclease variant or megaTAL and a polypeptide comprising an exonuclease. The term "exonuclease" refers to an enzyme that cleaves phosphodiester bonds at the end of a polynucleotide chain via a hydrolysis reaction that breaks the phosphodiester bond at either the 3' or 5' end.

[0295] Illustrative examples of exonucleases suitable for use in certain embodiments contemplated herein include, but are not limited to, hExoI, yeast ExoI, E. coli ExoI, hTREX2, mouse TREX2, rat TREX2, hTREX1, mouse TREX1, and rat TREX1.

[0296] In certain embodiments, the DNA end-processing enzyme is a 3' or 5' exonuclease, preferably Trex1 or Trex2, more preferably Trex2, even more preferably human or mouse Trex2.

[0297] D. Target site In certain embodiments, the contemplated nuclease variants can be designed to bind to any suitable target sequence and can have novel binding specificities compared to naturally occurring nucleases. In certain embodiments, the target site is a regulatory region of a gene, including but not limited to a promoter, enhancer, repressor element, etc. In certain embodiments, the target site is a coding region or splice site of a gene. In certain embodiments, the nuclease variant is designed to downregulate or reduce gene expression. In certain embodiments, the nuclease variant and the donor repair template can be designed to repair or delete a desired target sequence.

[0298] In various embodiments, the nuclease variant binds to and cleaves a target sequence within the human Casitas B-lineage (Cbl) lymphoma proto-oncogene B (CBLB) gene. CBL is also referred to as CBL2; Noonan syndrome-related disorder with or without juvenile myelomonocytic leukemia (NSLL); C-CBL; RING finger protein 55 (RNF55); fragile site, folate-type, rare, fra(11)(q23.3)(FRA11B); E3 ubiquitin protein ligase CBL; Cas-Br-M (murine) ecotropic retroviral transforming sequence; Cbl proto-oncogene, E3 ubiquitin protein ligase; RING-type E3 ubiquitin transferase CBL; Casitas B-lineage lymphoma proto-oncogene; oncogene CBL2; proto-oncogene c-Cbl; and signal transduction protein CBL.

[0299] This gene is a proto-oncogene that encodes a RING finger E3 ubiquitin ligase. The encoded protein is one of the enzymes required to target substrates for degradation by the proteasome. This protein mediates the transfer of ubiquitin from ubiquitin complexing enzymes (E2s) to specific substrates. This protein also contains an N-terminal phosphotyrosine-binding domain that enables it to interact with numerous tyrosine-phosphorylated substrates and target them for proteasomal degradation. CBLB functions as a negative regulator of many signaling pathways, including T cell activation and persistence.

[0300] In certain embodiments, the homing endonuclease variant or megaTAL introduces a double-stranded break (DSB) at a target site in the CBLB gene. In certain embodiments, the homing endonuclease variant or megaTAL introduces a DSB in exon 6 of the CBLB gene, preferably at SEQ ID NO: 20 in exon 6 of the CBLB gene, and more preferably at the sequence "ATTC" within SEQ ID NO: 20 in exon 6 of the CBLB gene.

[0301] In a preferred embodiment, the homing endonuclease variant or megaTAL cleaves double-stranded DNA and introduces a DSB into the polynucleotide sequence shown in SEQ ID NO: 20 or 22.

[0302] In a preferred embodiment, the CBLB gene is a human CBLB gene.

[0303] E. Donor repair template Nuclease variants can be used to introduce DSBs into target sequences, which can be repaired through the homology-directed repair (HDR) mechanism in the presence of one or more donor repair templates.

[0304] In various embodiments, the donor repair template comprises one or more polynucleotides encoding an immune enhancer, an immune suppressive signal damper, or an engineered antigen receptor.

[0305] In various embodiments, providing cells with engineered nucleases in the presence of multiple donor repair templates that independently encode immune enhancers and / or immune suppressive signal dampers targeting different immune suppressive pathways is contemplated to result in genome-edited T cells with increased therapeutic efficacy and persistence. For example, immune enhancers or immune suppressive signals targeting a combination of the PD-1, LAG-3, CTLA-4, TIM3, IL-10R, TIGIT, and TGFβRII pathways may be preferred in certain embodiments.

[0306] In certain embodiments, the donor repair template is used to insert a sequence into a genome. In certain preferred embodiments, the donor repair template is used to repair or modify a sequence within a genome.

[0307] In various embodiments, the donor repair template is introduced into hematopoietic cells, e.g., T cells, by transducing the cells with an adeno-associated virus (AAV), retrovirus, e.g., lentivirus, IDLV, herpes simplex virus, adenovirus, or vaccinia virus vector containing the donor repair template.

[0308] In certain embodiments, the donor repair template comprises one or more homology arms flanking the DSB site.

[0309] As used herein, the term "homologous arm" refers to a nucleic acid sequence in a donor repair template that is identical or nearly identical to the DNA sequence adjacent to the DNA break introduced by a nuclease at the target site. In one embodiment, the donor repair template includes a 5' homologous arm containing a nucleic acid sequence identical or nearly identical to the DNA sequence 5' at the DNA break site. In one embodiment, the donor repair template includes a 3' homologous arm containing a nucleic acid sequence identical or nearly identical to the DNA sequence 3' at the DNA break site. In a preferred embodiment, the donor repair template includes a 5' homologous arm and a 3' homologous arm. The donor repair template may contain homology to a genomic sequence immediately adjacent to the DSB site or to a genomic sequence within any number of base pairs from the DSB site. In certain embodiments, the pair of homologous arms includes a polynucleotide sequence containing a target site for a double-strand break with a mutation at the target site to minimize re-cutting of the target site. In one embodiment, the donor repair template comprises nucleic acid sequences homologous to a genomic sequence or homologous arms of about 5 bp, about 10 bp, about 25 bp, about 50 bp, about 100 bp, about 250 bp, about 500 bp, about 1000 bp, about 2500 bp, about 5000 bp, about 10000 bp or more, including any intervening length of homologous sequence.

[0310] Illustrative examples of suitable lengths of the homology arms contemplated in certain embodiments are 5 bp, about 10 bp, about 25 bp, about 50 bp, about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, which may be independently selected and include all intervening lengths of the homology arms. , about 1300 bp, about 1400 bp, about 1500 bp, about 1600 bp, about 1700 bp, about 1800 bp, about 1900 bp, about 2000 bp, about 2100 bp, about 2200 bp, about 2300 bp, about 2400 bp, about 2500 bp, about 2600 bp, about 2700 bp, about 2800 bp, about 2900 bp, or about 3000 bp or more.

[0311] Additional illustrative examples of suitable homology arm lengths include, but are not limited to, about 100 bp to about 3000 bp, about 200 bp to about 3000 bp, about 300 bp to about 3000 bp, about 400 bp to about 3000 bp, about 500 bp to about 3000 bp, about 500 bp to about 2500 bp, about 500 bp to about 2000 bp, about 750 bp to about 2000 bp, about 750 bp to about 1500 bp, or about 1000 bp to about 1500 bp, including all intervening lengths of the homology arms.

[0312] In certain embodiments, the lengths of the 5' and 3' homologous arms are independently selected from about 500 bp to about 1500 bp. In one embodiment, the 5' homologous arm is about 1500 bp and the 3' homologous arm is about 1000 bp. In one embodiment, the 5' homologous arm is about 200 bp to about 600 bp and the 3' homologous arm is about 200 bp to about 600 bp. In one embodiment, the 5' homologous arm is about 200 bp and the 3' homologous arm is about 200 bp. In one embodiment, the 5' homologous arm is about 300 bp and the 3' homologous arm is about 300 bp. In one embodiment, the 5' homologous arm is about 400 bp and the 3' homologous arm is about 400 bp. In one embodiment, the 5' homologous arm is about 500 bp and the 3' homologous arm is about 500 bp. In one embodiment, the 5' homologous arm is about 600 bp and the 3' homologous arm is about 600 bp.

[0313] In certain embodiments, the donor repair template may comprise one or more expression cassettes. In certain embodiments, the donor repair template may comprise one or more homologous arms and one or more polynucleotides, including, but not limited to, promoters and / or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, polynucleotides encoding self-cleaving polypeptides, and epitope tags.

[0314] In various embodiments, the donor repair template comprises a 5' homology arm, an RNA polymerase II promoter, one or more polynucleotides encoding an immunopotency enhancer, an immunosuppressive signal damper, or an engineered antigen receptor, and a 3' homology arm.

[0315] In various embodiments, the target site is modified with a donor repair template comprising a 5' homology arm and one or more polynucleotides encoding a self-cleaving viral peptide, e.g., T2A, an immune enhancer, an immunosuppressive signal damper, or an engineered antigen receptor, optionally a poly(A) signal or a self-cleaving peptide, and a 3' homology arm, wherein expression of the one or more polynucleotides is driven by the endogenous CBLB promoter.

[0316] 1. Immune Enhancer In certain embodiments, genome-edited immune effector cells contemplated herein are made more potent and / or resistant to immunosuppressive factors by introducing a DSB in the CBLB gene in the presence of a donor repair template comprising a polynucleotide encoding an immunopotentiator. As used herein, the term "immunopotentiator" refers to non-naturally occurring molecules that stimulate and / or enhance T cell activation and / or function, immunostimulatory factors, and non-naturally occurring polypeptides that convert immunosuppressive signals from the tumor microenvironment into immunostimulatory signals within T cells or other immune cells.

[0317] In certain embodiments, the immune enhancer is selected from the group consisting of a bispecific T cell engager (BiTE) molecule; an immunostimulatory factor, including but not limited to, a cytokine, a chemokine, a cytotoxin, and / or a cytokine receptor; and a flip receptor.

[0318] In some embodiments, the immunopotentiator, immunostimulator, or flip receptor is a fusion polypeptide that includes a protein destabilization domain.

[0319] a. Bispecific T cell engager (BiTE) molecules In certain embodiments, the genome-edited immune effector cells contemplated herein are made more potent by introducing a DSB in the CBLB gene in the presence of a donor repair template containing a polynucleotide encoding a bispecific T cell engager (BiTE) molecule. BiTE molecules are bipartite molecules comprising a first binding domain that binds to a target antigen, linker, or spacer, as contemplated elsewhere herein, and a second binding domain that binds to a stimulatory or costimulatory molecule on the immune effector cell. The first and second binding domains may be independently selected from a ligand, receptor, antibody or antigen-binding fragment thereof, lectin, and carbohydrate.

[0320] In certain embodiments, the first and second binding domains are antigen-binding domains.

[0321] In certain embodiments, the first and second binding domains are antibodies or antigen-binding fragments thereof. In one embodiment, the first and second binding domains are single-chain variable fragments (scFv).

[0322] Illustrative examples of target antigens that may be recognized and bound by the first binding domain in certain embodiments include alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, and the like. , fetal AchR, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM, VEGFR2, and WT-1.

[0323] Other exemplary embodiments of target antigens include MHC-peptide complexes, optionally wherein the peptide is selected from the group consisting of alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), Processed from the IL-1, EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, GD2, GD3, glypican-3 (GPC3), MAGE1, NY-ESO-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM, VEGFR2, and WT-1.

[0324] Illustrative examples of stimulatory or costimulatory molecules on immune effector cells that are recognized and bound by the second binding domain in certain embodiments include, but are not limited to, CD3γ, CD3δ, CD3ε, CD3ζ, CD28, CD134, CD137, and CD278.

[0325] In certain embodiments, a DSB is induced in the CBLB gene by an engineered nuclease, and a donor repair template comprising a polynucleotide encoding a BiTE is introduced into the cell and inserted into the CBLB gene by homologous recombination.

[0326] B immunostimulatory factors In certain embodiments, the genome-edited immune effector cells contemplated herein are made more potent by increasing immunostimulatory factors in either genome-edited cells or cells in the tumor microenvironment.Immune stimulatory factors refer to specific cytokines, chemokines, cytotoxins, and cytokine receptors that enhance immune responses in immune effector cells.In one embodiment, T cells are genetically engineered by introducing DSBs in the CBLB gene in the presence of a donor repair template containing a polynucleotide encoding a cytokine, chemokine, cytotoxin, or cytokine receptor.

[0327] In certain embodiments, the donor repair template comprises a polynucleotide encoding a cytokine selected from the group consisting of IL-2, insulin, IFN-γ, IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-α.

[0328] In certain embodiments, the donor repair template comprises a polynucleotide encoding a chemokine selected from the group consisting of MIP-1α, MIP-1β, MCP-1, MCP-3, and RANTES.

[0329] In certain embodiments, the donor repair template comprises a polynucleotide encoding a cytotoxin selected from the group consisting of perforin, granzyme A, and granzyme B.

[0330] In certain embodiments, the donor repair template comprises a polynucleotide encoding a cytokine receptor selected from the group consisting of an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, and an IL-21 receptor.

[0331] c. Flip receptor In certain embodiments, the genome-edited immune effector cells contemplated herein are engineered to be more resistant to exhaustion by "flipping" or "reversing" immunosuppressive signals induced by tumor microenvironment-induced immunosuppressive factors into positive immunostimulatory signals. In one embodiment, T cells are engineered by introducing a DSB in the CBLB gene in the presence of a donor repair template containing a polynucleotide encoding a flip receptor. As used herein, the term "flip receptor" refers to a non-naturally occurring polypeptide that converts immunosuppressive signals from the tumor microenvironment into immunostimulatory signals within T cells. In a preferred embodiment, a flip receptor refers to a polypeptide that includes an extracellular domain that binds to an immunosuppressive factor, a transmembrane domain, and an intracellular domain that transmits immunostimulatory signals to T cells.

[0332] In one embodiment, the donor repair template encodes a flip receptor that includes an extracellular domain or extracellular binding domain that binds an immunosuppressive cytokine, a transmembrane domain, and an intracellular domain of an immunostimulatory cytokine receptor.

[0333] In certain embodiments, the flip receptor comprises an extracellular domain that binds an immunosuppressive cytokine that is the extracellular cytokine binding domain of IL-4 receptor, IL-6 receptor, IL-8 receptor, IL-10 receptor, IL-13 receptor, TGFβ receptor 1, or TGFβ receptor 2; a transmembrane domain isolated from CD4, CD8α, CD27, CD28, CD134, CD137, CD3, TGFβ receptor 1, TGFβ receptor 2, IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, or IL-21 receptor; and an intracellular domain isolated from IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, or IL-21 receptor.

[0334] In certain embodiments, the flip receptor comprises an extracellular domain that binds an immunosuppressive cytokine that is an antibody or antigen-binding fragment thereof that binds to IL-4, IL-6, IL-8, IL-10, IL-13, TGFβ receptor 1, or TGFβ receptor 2; a transmembrane domain isolated from CD4, CD8α, CD27, CD28, CD134, CD137, CD3, TGFβ receptor 1, or TGFβ receptor 2, IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, or IL-21 receptor; and an intracellular domain isolated from IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, or IL-21 receptor.

[0335] In one embodiment, the donor repair template comprises a flip receptor comprising an extracellular domain that binds to an immunosuppressive factor, a transmembrane domain, and one or more intracellular costimulatory signaling domains and / or primary signaling domains.

[0336] Illustrative examples of extracellular domains suitable for use in certain embodiments of contemplated flip receptors include, but are not limited to, the extracellular ligand-binding domains of receptors that contain ITIMs and / or ITSMs.

[0337] Further illustrative examples of extracellular domains suitable for use in certain embodiments of contemplated flip receptors include, but are not limited to, the extracellular ligand-binding domains of PD-1, LAG-3, TIM-3, CTLA-4, BTLA, CEACAM1, TIGIT, TGFβRI, TGFβRII, IL4R, IL6R, CXCR1, CXCR2, IL10R, IL13Rα2, TRAILR1, RCAS1R, and FAS.

[0338] In one embodiment, the extracellular domain comprises the extracellular ligand-binding domain of a receptor selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, IL10R, TIGIT, TGFβRI, and TGFβRII.

[0339] In one embodiment, the donor repair template comprises a flip receptor comprising an extracellular domain that binds to an immunosuppressive cytokine, a transmembrane domain, and one or more intracellular costimulatory signaling domains and / or primary signaling domains.

[0340] Illustrative examples of membrane spanning domains suitable for use in certain embodiments of the flip receptors contemplated in certain embodiments include, but are not limited to, the alpha or beta chain transmembrane domains of the following proteins: PD-1, LAG-3, TIM-3, CTLA-4, IL10R, TIGIT, TGFβRI, and TGFβRII T-cell receptors, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, or CD154.

[0341] In various embodiments, the flip receptor comprises an intracellular domain that induces an immunostimulatory signal. As used herein, the term "intracellular domain" refers to an immunostimulatory motif or domain, including, but not limited to, an immunoreceptor tyrosine-based activation motif (ITAM), a costimulatory signaling domain, a primary signaling domain, or another intracellular domain associated with inducing an immunostimulatory signal in a T cell.

[0342] Illustrative examples of intracellular domains suitable for use in certain embodiments of the flip receptors contemplated in certain embodiments include, but are not limited to, domains containing ITAM motifs.

[0343] Additional illustrative examples of intracellular domains suitable for use in certain embodiments of the flip receptors contemplated in certain embodiments include, but are not limited to, costimulatory signaling domains isolated from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, or ZAP70.

[0344] Additional illustrative examples of intracellular domains suitable for use in certain embodiments of contemplated flip receptors include, but are not limited to, intracellular domains isolated from IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, or IL-21 receptor.

[0345] Further illustrative examples of intracellular domains suitable for use in certain embodiments of contemplated flip receptors include, but are not limited to, the major signaling domains isolated from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0346] In certain embodiments, the flip receptor comprises an extracellular domain comprising an extracellular domain from PD-1, LAG-3, TIM-3, CTLA-4, IL10R, TIGIT, TGFβRI, or TGFβRII, and an intracellular domain isolated from an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, or an IL-21 receptor.

[0347] In certain embodiments, the flip receptor comprises an extracellular domain comprising an extracellular domain from PD-1, LAG-3, TIM-3, CTLA-4, IL10R, TIGIT, TGFβRI, or TGFβRII, a transmembrane domain from a CD3 polypeptide, CD4, CD8α, CD28, CD134, CD137, PD-1, LAG-3, TIM-3, CTLA-4, IL10R, TGFβRI, and TGFβRII, and an intracellular domain from CD28, CD134, CD137, CD278, and / or CD3ζ.

[0348] In certain embodiments, the flip receptor comprises an extracellular domain comprising an extracellular domain from PD-1, LAG-3, TIM-3, CTLA-4, IL10R, TIGIT, or TGFβRII, a transmembrane domain from a CD3 polypeptide, CD4, CD8α, CD28, CD134, or CD137, and an intracellular domain from CD28, CD134, CD137, CD278, and / or CD3ζ.

[0349] 2. Immunosuppressive signal dampers One limitation or problem plaguing existing adoptive cell therapy is the hyporesponsiveness of immune effector cells due to exhaustion mediated by the tumor microenvironment. Exhausted T cells have a unique molecular signature that is significantly different from naive, effector, or memory T cells. They are defined as T cells with reduced cytokine expression and effector function.

[0350] In certain embodiments, the genome-edited immune effector cells contemplated herein are made more resistant to exhaustion by reducing or attenuating signaling by immunosuppressive factors. In one embodiment, T cells are engineered by introducing a DSB in the CBLB gene in the presence of a donor repair template comprising a polynucleotide encoding an immunosuppressive signal damper.

[0351] As used herein, the term "immunosuppressive signal damper" refers to a non-naturally occurring polypeptide that reduces the transmission of immunosuppressive signals from the tumor microenvironment to T cells. In one embodiment, the immunosuppressive signal damper is an antibody or antigen-binding fragment thereof that binds to an immunosuppressive factor. In a preferred embodiment, the immunosuppressive signal damper refers to a polypeptide that suppresses, represses, or induces a dominant-negative effect on a specific immunosuppressive factor or signaling pathway because the damper comprises an extracellular domain that binds to the immunosuppressive factor, optionally a transmembrane domain, and optionally a modified intracellular domain (e.g., an intracellular signaling domain).

[0352] In certain embodiments, the extracellular domain is an extracellular binding domain that recognizes and binds to an immunosuppressive factor.

[0353] In certain embodiments, the modified intracellular domain is mutated to reduce or inhibit immunosuppressive signaling. Suitable mutation strategies include, but are not limited to, amino acid substitution, addition, or deletion. Suitable mutations also include, but are not limited to, truncating the intracellular domain to remove the signaling domain, mutating the intracellular domain to remove residues important for signaling motif activity, and mutating the intracellular domain to block receptor cycling. In certain embodiments, the intracellular domain, when present, does not transmit immunosuppressive signals or has substantially reduced signaling.

[0354] Thus, in some embodiments, the immunosuppressive signal dampener acts as a sink for one or more immunosuppressive factors from the tumor microenvironment and inhibits the corresponding immunosuppressive signaling pathway in T cells.

[0355] One immunosuppressive signal is mediated by tryptophan catabolism.Tryptophan catabolism by indoleamine 2,3-dioxygenase (IDO) in cancer cells leads to the production of kynurenine, which has been shown to have immunosuppressive effects on T cells in tumor microenvironment.See, for example, Platten et al.(2012)Cancer Res.72(21):5435-40.

[0356] In one embodiment, the donor repair template comprises an enzyme with kynureninase activity.

[0357] Illustrative examples of enzymes having kynureninase activity suitable for use in certain embodiments include, but are not limited to, L-kynurenine hydrolase.

[0358] In one embodiment, the donor repair template comprises one or more polynucleotides encoding an immunosuppressive signal damper that reduces or blocks immunosuppressive signaling mediated by an immunosuppressive factor.

[0359] Illustrative examples of immunosuppressive factors targeted by immunosuppressive signal dampers contemplated in certain embodiments include, but are not limited to, programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), transforming growth factor beta (TGFβ), macrophage colony-stimulating factor 1 (M-CSF1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), receptor-associated cancer antigen expressed on SiSo cells ligand (RCAS1), Fas ligand (FasL), CD47, interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), and interleukin-13 (IL-13).

[0360] In various embodiments, the immunosuppressive signal damper comprises an antibody or antigen-binding fragment thereof that binds to the immunosuppressive factor.

[0361] In various embodiments, the immunosuppressive signal damper comprises an extracellular domain that binds to an immunosuppressive factor.

[0362] In certain embodiments, the immunosuppressive signal damper comprises an extracellular domain that binds to an immunosuppressive factor and a transmembrane domain.

[0363] In another embodiment, the immunosuppressive signal damper comprises an extracellular domain that binds to an immunosuppressive factor, a transmembrane domain, and a modified intracellular domain that does not transmit an immunosuppressive signal or has a substantially reduced ability to transmit an immunosuppressive signal.

[0364] As used herein, the term "extracellular domain" refers to an antigen-binding domain. In one embodiment, the extracellular domain is the extracellular ligand-binding domain of an immunoinhibitory receptor that transmits immunosuppressive signals from the tumor microenvironment to T cells. In a specific embodiment, the extracellular domain refers to the extracellular ligand-binding domain of a receptor that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and / or an immunoreceptor tyrosine-based switch motif (ITSM).

[0365] Illustrative examples of extracellular domains suitable for use in certain embodiments of immunosuppressive signal dampers include antibodies or antigen-binding fragments thereof, or the following polypeptides: programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 protein (LAG-3), T-cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuating factor (BTLA), T-cell immunoglobulin and immunoreceptor tyrosine-dependent inhibitory motif domain (TIGIT), transforming growth factor beta receptor 1 (TGFβRI), transforming growth factor beta receptor 2 (TGFβRII), macrophage. These include, but are not limited to, the extracellular ligand-binding domain isolated from phage colony-stimulating factor 1 receptor (CSF1R), interleukin 4 receptor (IL4R), interleukin 6 receptor (IL6R), chemokine (C-X-C motif) receptor 1 (CXCR1), chemokine (C-X-C motif) receptor 2 (CXCR2), interleukin 10 receptor subunit alpha (IL10R), interleukin 13 receptor subunit alpha 2 (IL13Rα2), tumor necrosis factor-related apoptosis-inducing ligand (TRAILR1), receptor-binding cancer antigen expressed on SiSo cells (RCAS1R), and the Fas cell surface death receptor (FAS).

[0366] In one embodiment, the extracellular domain comprises the extracellular ligand-binding domain of a receptor selected from the group consisting of PD-1, LAG-3, TIM3, CTLA-4, IL10R, TIGIT, CSF1R, TGFβRII, and TGFβRII.

[0367] Many transmembrane domains can be used in certain embodiments. Illustrative examples of transmembrane domains suitable for use in certain embodiments of the immunosuppressive signal dampers contemplated in certain embodiments include, but are not limited to, the transmembrane domains of the alpha or beta chains of the following proteins: T-cell receptor, CD5, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD-1.

[0368] 3. Genetically engineered antigen receptors In certain embodiments, the genome-edited immune effector cells contemplated herein comprise a genetically engineered antigen receptor. In one embodiment, T cells comprising a polynucleotide encoding the genetically engineered antigen receptor are edited by introducing a DSB in the CBLB gene. In one embodiment, T cells are genetically engineered by introducing a DSB in the CBLB gene in the presence of a donor repair template comprising a polynucleotide encoding the genetically engineered antigen receptor.

[0369] In certain embodiments, the engineered antigen receptor is an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a Daric receptor or a component thereof, or a chimeric cytokine receptor.

[0370] a. Genetically engineered TCR In certain embodiments, the genome-edited immune effector cells contemplated herein comprise a polynucleotide encoding a genetically engineered TCR. In one embodiment, T cells comprising a polynucleotide encoding a genetically engineered TCR are edited by introducing a DSB in the CBLB gene. In one embodiment, T cells are engineered by introducing a DSB in the CBLB gene in the presence of a donor repair template encoding the genetically engineered TCR. In certain embodiments, the genetically engineered TCR is inserted at a DSB in a single CBLB allele. In another embodiment, the alpha chain of the genetically engineered TCR is inserted at a DSB in one CBLB allele, and the beta chain of the genetically engineered TCR is inserted at a DSB in the other CBLB allele.

[0371] In one embodiment, the engineered T cells contemplated herein comprise a polynucleotide encoding an engineered TCR that is not inserted at a CBLB gene, and / or one or more of an immunosuppressive signal damper, a flip receptor, an engineered alpha and / or beta chain of a T cell receptor (TCR), a chimeric antigen receptor (CAR), a Daric receptor or component thereof, or a chimeric cytokine receptor is inserted at a DSB in one or more CBLB genes.

[0372] Naturally occurring T cell receptors contain two subunits, alpha and beta chains, each of which is a unique protein produced by recombination in the genome of each T cell. A library of TCRs can be screened for their selectivity for specific target antigens. In this way, natural TCRs with high avidity and reactivity to target antigens can be selected, cloned, and then introduced into a population of T cells used for adoptive immunotherapy.

[0373] In one embodiment, T cells are modified by introducing a donor repair template containing a polynucleotide encoding a TCR subunit with a DSB in one or more CBLB alleles, where the TCR subunit has the ability to form a TCR that confers specificity to the T cell against tumor cells expressing a target antigen. In certain embodiments, the subunit has one or more amino acid substitutions, deletions, insertions, or modifications compared to the naturally occurring subunit, so long as the subunit retains the ability to form a TCR, confers the transfected T cell's ability to be directed to target cells, and participates in immunologically relevant cytokine signaling. The engineered TCR also preferably binds to target cells displaying relevant tumor-associated peptides with high activity and, optionally, mediates efficient killing of target cells presenting the relevant peptide in vivo.

[0374] The nucleic acids encoding the engineered TCRs are preferably isolated from their natural context in the (naturally occurring) chromosomes of T cells and can be incorporated into a suitable vector, as described elsewhere herein. Both the nucleic acids and the vectors containing them can be transferred into cells, preferably, in certain embodiments, into T cells. The modified T cells can then express one or more chains of the TCR encoded by the transduced nucleic acid(s). In a preferred embodiment, the engineered TCR is an exogenous TCR because it is introduced into T cells that do not normally express a specific TCR. An essential aspect of an engineered TCR is that it has high avidity for tumor antigens presented by major histocompatibility complexes (MHC) or similar immunological components. In contrast to engineered TCRs, CARs are engineered to bind to target antigens in an MHC-independent manner.

[0375] The TCR may be expressed with additional polypeptides attached to the amino- or carboxyl-terminal portions of the alpha or beta chain of the TCR of the present invention, so long as the attached additional polypeptides do not interfere with the ability of the alpha or beta chain to form a functional T cell receptor and MHC-dependent antigen recognition.

[0376] Antigens recognized by engineered TCRs contemplated in certain embodiments include, but are not limited to, cancer antigens, including antigens in both hematological cancers and solid tumors. Exemplary antigens include alpha-folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, GD2, GD3, and glypican-3. These include, but are not limited to, HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM, VEGFR2, and WT-1.

[0377] In one embodiment, the donor repair template comprises a polynucleotide encoding an RNA polymerase II promoter or a first self-cleaving viral peptide and a polynucleotide encoding the alpha and / or beta chain of an engineered TCR integrated into one modified and / or dysfunctional CBLB gene.

[0378] In one embodiment, the donor repair template comprises a polynucleotide encoding an RNA polymerase II promoter or a first self-cleaving viral peptide and polynucleotides encoding the alpha and beta chains of an engineered TCR integrated into one modified and / or dysfunctional CBLB gene.

[0379] In certain embodiments, the donor repair template comprises a 5'-3' polynucleotide encoding a first self-cleaving viral peptide, a polynucleotide encoding the alpha chain of an engineered TCR, a polynucleotide encoding a second self-cleaving viral peptide, and a polynucleotide encoding the beta chain of an engineered TCR integrated into one modified and / or dysfunctional CBLB gene. In such cases, the other CBLB gene may be functional or may be rendered functionally impaired or dysfunctional by a DSB and repaired by NHEJ. In one embodiment, the other CBLB gene is modified and rendered functionally impaired or dysfunctional by an engineered nuclease contemplated herein.

[0380] In certain embodiments, both CBLB genes are modified to have reduced or no function, wherein the first modified CBLB gene comprises a nucleic acid comprising a polynucleotide encoding a first self-cleaving viral peptide and a polynucleotide encoding the alpha chain of an engineered TCR, and the second modified CBLB gene comprises a polynucleotide encoding a second self-cleaving viral peptide and a polynucleotide encoding the beta chain of an engineered TCR.

[0381] b. Chimeric Antigen Receptor (CAR) In certain embodiments, the genetically engineered immune effector cells contemplated herein comprise one or more chimeric antigen receptors (CARs). In one embodiment, T cells comprising a polynucleotide encoding one or more CARs are edited by introducing a DSB in a CBLB gene. In one embodiment, T cells are genetically engineered by introducing a DSB in one or more CBLB genes in the presence of a donor repair template encoding a CAR. In certain embodiments, a CAR is inserted at a DSB in a single CBLB gene.

[0382] In one embodiment, the engineered T cells contemplated herein, a CAR not inserted in a CBLB gene, and one or more of an immunosuppressive signal damper, a flip receptor, an engineered alpha and / or beta chain of a T cell receptor (TCR), a chimeric antigen receptor (CAR), a Daric receptor or component thereof, or a chimeric cytokine receptor are inserted into a DSB in one or more CBLB genes.

[0383] In various embodiments, the genome-edited T cells express a CAR that redirects cytotoxicity against tumor cells. A CAR is a molecule that combines antibody-based specificity for a target antigen (e.g., a tumor antigen) with an intracellular domain that activates a T cell receptor to generate a chimeric protein that exhibits specific anti-tumor cell-mediated immune activity. As used herein, the term "chimeric" describes a protein composed of parts of different proteins or DNA from different sources.

[0384] In various embodiments, CARs comprise an extracellular domain that binds to a specific target antigen (also referred to as a binding domain or antigen-specific binding domain), a transmembrane domain, and an intracellular signaling domain. A key property of CARs is their ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis, or production of molecules that can mediate cell death of cells expressing the target antigen in a major histocompatibility (MHC)-independent manner, utilizing the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors.

[0385] In certain embodiments, the CAR comprises an extracellular binding domain that specifically binds to a target polypeptide, for example, a target antigen expressed on a tumor cell. As used herein, the terms "binding domain," "extracellular domain," "extracellular binding domain," "antigen binding domain," "antigen-specific binding domain," and "extracellular antigen-specific binding domain" are used interchangeably to provide a chimeric receptor, for example, a CAR or Daric, capable of specifically binding to a target antigen of interest. The binding domain can include any protein, polypeptide, oligopeptide, or peptide that has the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule or component thereof). The binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for the biological molecule of interest.

[0386] In certain embodiments, the extracellular binding domain comprises an antibody or an antigen-binding fragment thereof.

[0387] "Antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of a target antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant, e.g., one recognized by immune cells. Antibodies include antigen-binding fragments, such as camelid Ig (camelid antibody or VHH fragment thereof), Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single-chain Fv antibodies ("scFv"), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv"), and single-domain antibodies (sdAb, Nanobody) or other antibody fragments. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

[0388] In one preferred embodiment, the binding domain is an scFv.

[0389] In another preferred embodiment, the binding domain is a camelid antibody.

[0390] In certain embodiments, the CAR is selected from the group consisting of alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, GD2, GD3, glypican-3 (GPC3), comprising an extracellular domain that binds to an antigen selected from the group consisting of HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM, VEGFR2, and WT-1.

[0391] In certain embodiments, the CAR comprises an extracellular binding domain, e.g., an antibody or antigen-binding fragment thereof, that binds to an antigen, where the antigen is an MHC-peptide complex, such as a class I MHC-peptide complex or a class II MHC-peptide complex.

[0392] In certain embodiments, a CAR contains linker residues between the various domains. A "variable region linking sequence" is an amino acid sequence that connects the heavy chain variable region to the light chain variable region and provides a spacer function compatible with the interaction of the two sub-binding domains so that the resulting polypeptide retains specific binding affinity for the same target molecule as an antibody comprising the same light and heavy chain variable regions. In certain embodiments, a CAR contains one, two, three, four, or five or more linkers. In certain embodiments, the linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids in length, or any intervening length of amino acids. In some embodiments, the linker is 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, or more amino acids in length.

[0393] In certain embodiments, the binding domain of the CAR is followed by one or more "spacer domains," which refer to regions that separate the antigen-binding domain from the effector cell surface to allow proper cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). Spacer domains can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0394] In one embodiment, the spacer domain comprises CH2 and CH3 of IgG1, IgG4, or IgD.

[0395] In one embodiment, the binding domain of the CAR is linked to one or more "hinge domains" that serve to position the antigen-binding domain away from the effector cell surface, allowing for proper cell-cell contact, antigen binding, and activation. CARs generally contain one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0396] Exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8α and CD4, which may be wild-type hinge regions from these molecules or may be modified. In another embodiment, the hinge domain comprises a CD8α hinge region.

[0397] In one embodiment, the hinge is a PD-1 hinge or a CD152 hinge.

[0398] The "transmembrane domain" is the portion of the CAR that fuses the extracellular binding moiety and the intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell. The TM domain can be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0399] Exemplary TM domains can be derived from (i.e., can include) at least the transmembrane region(s) of the alpha or beta chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PD-1.

[0400] In one embodiment, the CAR comprises a TM domain derived from CD8α. In another embodiment, the CAR contemplated herein comprises a TM domain derived from CD8α and a short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the TM domain and the intracellular signaling domain of the CAR. A glycine-serine linker provides a particularly suitable linker.

[0401] In certain embodiments, the CAR comprises an intracellular signaling domain. "Intracellular signaling domain" refers to the portion of the CAR that is involved in transmitting the message of effective CAR binding to a target antigen inside an immune effector cell to elicit effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors into the CAR-bound target cell, or other cellular responses elicited by antigen binding to the extracellular CAR domain.

[0402] The term "effector function" refers to a specialized function of a cell. Effector function of a T cell can be, for example, cytolytic or adjuvant activity, or activity including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and directs the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, it can be used in place of the entire domain, so long as such truncated portion transmits the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0403] It is known that the signal generated by TCR alone is insufficient for the complete activation of T cells, and that secondary or costimulatory signals are also required.Therefore, it can be said that T cell activation is mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation by TCR (e.g., TCR / CD3 complex), and costimulatory signaling domains that act in an antigen-independent manner to provide secondary or costimulatory signals.In a preferred embodiment, CAR comprises an intracellular signaling domain that comprises one or more "costimulatory signaling domains" and "primary signaling domains".

[0404] The primary signaling domain regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. A stimulatory primary signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM.

[0405] Illustrative examples of ITAMs containing a major signaling domain suitable for use in CARs contemplated in certain embodiments include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In particularly preferred embodiments, the CAR comprises a CD3ζ major signaling domain and one or more costimulatory signaling domains. The intracellular major signaling domain and the costimulatory signaling domain can be linked in tandem to the carboxyl terminus of the transmembrane domain in any order.

[0406] In certain embodiments, the CAR comprises one or more costimulatory signaling domains to enhance the efficacy and expansion of T cells expressing the CAR receptor. As used herein, the term "costimulatory signaling domain," or "costimulatory domain," refers to the intracellular signaling domain of a costimulatory molecule.

[0407] Illustrative examples of such costimulatory molecules suitable for use in contemplated CARs in certain embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. In one embodiment, the CAR comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3ζ major signaling domain.

[0408] In various embodiments, the CAR comprises an extracellular domain that binds to an antigen selected from the group consisting of BCMA, CD19, CSPG4, PSCA, ROR1, and TAG72; a transmembrane domain isolated from a polypeptide selected from the group consisting of CD4, CD8α, CD154, and PD-1; one or more intracellular costimulatory signaling domains isolated from a polypeptide selected from the group consisting of CD28, CD134, and CD137; and a signaling domain isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0409] c.Daric receptor In certain embodiments, the engineered immune effector cells contain one or more Daric receptors. As used herein, the term "Daric receptor" refers to a multi-chain engineered antigen receptor. In one embodiment, T cells containing a polynucleotide encoding one or more Daric receptors are edited by introducing a DSB in a CBLB gene. In one embodiment, T cells are engineered by introducing a DSB in one or more CBLB alleles in the presence of a donor repair template encoding one or more components of Daric. In certain embodiments, Daric or one or more components thereof are inserted at a DSB in a single CBLB allele.

[0410] In one embodiment, the engineered T cells comprise Daric that is not inserted in a CBLB gene and one or more of an immunosuppressive signal damper, a flip receptor, an engineered alpha and / or beta chain of a T cell receptor (TCR), a chimeric antigen receptor (CAR), or the Daric receptor or a component thereof is inserted into a DSB in one or more CBLB alleles.

[0411] Illustrative examples of Daric structures and components are disclosed in PCT Publication WO2015 / 017214 and U.S. Patent Publication No. 20150266973, each of which is incorporated herein by reference in its entirety.

[0412] In one embodiment, the donor repair template comprises the following Daric components: a signaling polypeptide comprising a first multimerization domain, a first transmembrane domain, and one or more intracellular costimulatory signaling domains and / or a primary signaling domain, and a binding polypeptide comprising a binding domain, a second multimerization domain, and optionally, a second transmembrane domain. Functional Daric comprises a cross-linking factor associated with and positioned between the multimerization domains of the signaling polypeptide and the binding polypeptide, promoting the formation of a Daric receptor complex on the cell surface.

[0413] In certain embodiments, the first and second multimerization domains are associated with a cross-linking agent selected from the group consisting of rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, and any combination thereof.

[0414] Illustrative examples of rapamycin analogs (rapalogs) include those disclosed in U.S. Patent No. 6,649,595, the rapalog structures of which are incorporated herein by reference in their entirety. In certain embodiments, the cross-linking agent is a rapalog that has a substantially reduced immunosuppressive effect compared to rapamycin. A "substantially reduced immunosuppressive effect" refers to a rapalog that has at least 0.1 to 0.005 times the immunosuppressive effect observed or expected for an equimolar amount of rapamycin, as measured clinically in a softbank or suitable in vitro (e.g., inhibition of T-cell proliferation) or in vivo surrogate for human immunosuppressive activity. In one embodiment, a "substantially reduced immunosuppressive effect" refers to a rapalog that has an EC50 value in such an in vitro assay that is at least 10 to 250 times greater than the EC50 value observed for rapamycin in the same assay.

[0415] Other illustrative examples of rapalogs include, but are not limited to, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0416] In certain embodiments, the multimerization domain will be associated with a cross-linking agent that is rapamycin or its rapalog.For example, the first and second multimerization domains are a pair selected from FKBP and FRB.FRB domain is a polypeptide region (protein "domain") that can form a ternary complex with FKBP protein and rapamycin or its rapalog.FRB domain exists in many naturally occurring proteins, including mTOR protein (also referred to in literature as FRAP, RAPT1, or RAFT), yeast proteins including Tor1 and Tor2, and Candida FRAP homologues from humans and other species.The information on nucleotide sequence, cloning, and other aspects of these proteins has already been known in the art.For example, the protein sequence accession number for human mTOR is GenBank accession number L34075.1 (Brown et al., Nature 369:756, 1994).

[0417] FRB domains suitable for use in certain embodiments contemplated herein generally contain at least about 85 to about 100 amino acid residues. In certain embodiments, the FRB amino acid sequence used in the fusion proteins of the present disclosure will contain the 93 amino acid sequence Ile-2021 to Lys-2113 and the T2098L mutation, based on the amino acid sequence of GenBank Accession No. L34075.1. In certain embodiments, FRB domains for use in Daric contemplated will be capable of binding to a complex of an FKBP protein bound to rapamycin or a rapalog thereof. In certain embodiments, the peptide sequence of the FRB domain includes (a) a naturally occurring peptide sequence spanning at least the designated 93 amino acid region or corresponding region of the homologous protein human mTOR, (b) a naturally occurring variant of FRB in which up to about 10 amino acids, or about 1 to about 5 amino acids, or about 1 to about 3 amino acids, or in some embodiments, as few as one amino acid, of the naturally occurring peptide have been deleted, inserted, or substituted, or (c) a peptide encoded by a nucleic acid molecule capable of selectively hybridizing to a DNA molecule encoding a naturally occurring FRB domain, or by a DNA sequence that would be capable of selectively hybridizing to a DNA molecule encoding a naturally occurring FRB domain if there were no degeneracy in the genetic code.

[0418] FKBP (FK506-binding protein) is a cytoplasmic receptor for macrolides such as FK506, FK520, and rapamycin, and is highly conserved across species. FKBP is a protein or protein domain that can bind to rapamycin or its rapalogs and further form a tripartite complex with a protein or fusion protein containing FKBP. An FKBP domain may also be referred to as a "rapamycin-binding domain." Information related to the nucleotide sequences, cloning, and other aspects of various FKBP species is known in the art (see, e.g., Staendart et al., Nature 346:671, 1990 (human FKBP12); Kay, Biochem. J. 314:361, 1996). Homologous FKBP proteins in other mammalian species, yeast, and other organisms are also known in the art and can be used in the fusion proteins disclosed herein. In certain embodiments, contemplated FKBP domains will be capable of binding to rapamycin or a rapalog thereof and participating in a tripartite complex with a protein containing an FRB (as can be determined directly or indirectly by any means to detect such binding).

[0419] Illustrative examples of FKBP domains suitable for use in Daric contemplated in certain embodiments include, but are not limited to, peptide sequences preferably from or isolated from the human FKBP12 protein (GenBank Accession No. AAA58476.1); naturally occurring FKBP peptide sequences isolated from another human FKBP, from a mouse or other mammalian FKBP, or from some other animal, yeast, or fungal FKBP; variants of naturally occurring FKBPs in which up to about 10 amino acids, or from about 1 to about 5 amino acids, or from about 1 to about 3 amino acids, or in some embodiments, as few as one amino acid, of a naturally occurring peptide have been deleted, inserted, or substituted; or peptides encoded by nucleic acid molecules capable of selectively hybridizing to DNA molecules encoding naturally occurring FKBP domains, or by DNA sequences that would be capable of selectively hybridizing to DNA molecules encoding naturally occurring FKBPs if there were no degeneracy in the genetic code.

[0420] Other illustrative examples of multimerization domain pairs suitable for use in Daric contemplated in certain embodiments include, but are not limited to, FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof.

[0421] In yet another embodiment, the anti-crosslinking agent blocks the association of the signaling polypeptide and the binding polypeptide with the crosslinking agent. For example, cyclosporine or FK506 can be used as an anti-crosslinking agent to titrate rapamycin, thus stopping signal transduction because only one multimerization domain binds. In certain embodiments, the anti-crosslinking agent (e.g., cyclosporine, FK506) is an immunosuppressant. For example, an immunosuppressive anti-crosslinking agent can be used in certain embodiments to block or minimize the function of the intended Daric component, while simultaneously inhibiting or blocking undesirable or pathological inflammatory responses in clinical settings.

[0422] In one embodiment, the first multimerization domain comprises FRB T2098L, the second multimerization domain comprises FKBP12, and the cross-linking agent is the rapalog AP21967.

[0423] In another embodiment, the first multimerization domain comprises FRB, the second multimerization domain comprises FKBP12, and the cross-linking agent is rapamycin, temsirolimus, or everolimus.

[0424] In certain embodiments, the signaling polypeptide, the first transmembrane domain, and the binding polypeptide comprise a second transmembrane domain or a GPI anchor. Illustrative examples of the first and second transmembrane domains are isolated from polypeptides independently selected from the group consisting of CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PD-1.

[0425] In one embodiment, the signaling polypeptide comprises one or more intracellular costimulatory signaling domains and / or primary signaling domains.

[0426] Illustrative examples of major signaling domains suitable for use in Daric signaling components contemplated in certain embodiments include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In certain preferred embodiments, the Daric signaling component comprises a CD3ζ major signaling domain and one or more costimulatory signaling domains. The intracellular major signaling domain and the costimulatory signaling domain can be linked in tandem, in any order, to the carboxyl terminus of the transmembrane domain.

[0427] Illustrative examples of such costimulatory molecules suitable for use in Daric signaling components contemplated in certain embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. In one embodiment, the Daric signaling component comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3ζ major signaling domain.

[0428] In certain embodiments, the Daric binding entity comprises a binding domain, hi one embodiment, the binding domain is an antibody or antigen-binding fragment thereof.

[0429] An antibody or antigen-binding fragment thereof comprises at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of a target antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant, e.g., one recognized by an immune cell. Antibodies also include antigen-binding fragments, such as camel Ig (camelid antibody or VHH fragment thereof), Ig The term includes NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single-chain Fv antibodies ("scFv"), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv"), and single-domain antibodies (sdAb, Nanobody) or other antibody fragments. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

[0430] In one preferred embodiment, the binding domain is an scFv.

[0431] In another preferred embodiment, the binding domain is a camelid antibody.

[0432] In certain embodiments, the Daric binding entity is selected from the group consisting of alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, GD2, GD3, and Glypin. The antibody comprises an extracellular domain that binds to an antigen selected from the group consisting of Kan-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM, VEGFR2, and WT-1.

[0433] In one embodiment, the Daric binding component is an extracellular domain, e.g., class I It includes antibodies or antigen-binding fragments thereof that bind to MHC-peptide complexes, such as MHC-peptide complexes or class II MHC-peptide complexes.

[0434] In certain embodiments, the Daric components contemplated herein include a linker or spacer connecting two proteins, polypeptides, peptides, domains, regions, or motifs. In certain embodiments, the linker comprises about 2 to about 35 amino acids, or about 4 to about 20 amino acids, or about 8 to about 15 amino acids, or about 15 to about 25 amino acids. In other embodiments, the spacer may have a specific structure, such as an antibody CH2-CH3 domain, hinge domain, or the like. In one embodiment, the spacer comprises the CH2 and CH3 domains of IgG1, IgG4, or IgD.

[0435] In certain embodiments, Daric components contemplated herein contain one or more "hinge domains" that serve to position the domains to allow proper cell-cell contact, antigen binding, and activation. Daric may contain one or more hinge domains between the binding domain and the multimerization domain and / or transmembrane domain (TM), or between the multimerization domain and the transmembrane domain. The hinge domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. In certain embodiments, the hinge is a CD8α hinge or a CD4 hinge.

[0436] In one embodiment, Daric comprises a signaling polypeptide comprising a first multimerization domain of FRB T2098L, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ major signaling domain; a binding polypeptide comprising an scFv that binds CD19, a second multimerization domain of FKBP12, and a CD4 transmembrane domain; and a cross-linking agent is the rapalog AP21967.

[0437] In one embodiment, Daric comprises a signaling polypeptide comprising a first multimerization domain of FRB, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ major signaling domain; a binding polypeptide comprising an scFv that binds to CD19, a second multimerization domain of FKBP12, and a CD4 transmembrane domain; and a cross-linking agent is rapamycin, temsirolimus, or everolimus.

[0438] d. Zetakine In certain embodiments, the genetically engineered immune effector cells contemplated herein comprise one or more chimeric cytokine receptors. In one embodiment, T cells comprising a polynucleotide encoding zetakine are edited by introducing a DSB in the CBLB gene. In one embodiment, T cells are genetically engineered by introducing a DSB in one or more CBLB alleles in the presence of a donor repair template encoding a CAR. In certain embodiments, the chimeric cytokine receptor is inserted at a DSB in a single CBLB gene.

[0439] In one embodiment, the engineered T cells contemplated herein, the chimeric cytokine receptor not inserted in a CBLB gene, and one or more of an immunosuppressive signal damper, a flip receptor, an engineered alpha and / or beta chain of a T cell receptor (TCR), a chimeric antigen receptor (CAR), a Daric receptor or component thereof, or a chimeric cytokine receptor are inserted into a DSB in one or more CBLB genes.

[0440] In various embodiments, genome-edited T cells express chimeric cytokine receptors that redirect cytotoxicity against tumor cells. Zetakines are chimeric transmembrane immunoreceptors that include an extracellular domain containing a soluble receptor ligand linked to a support region capable of linking the extracellular domain to the cell surface, a transmembrane region, and an intracellular signaling domain. When expressed on the surface of T lymphocytes, zetakines direct T cell activity to cells expressing receptors specific for the soluble receptor ligand. Zetakine chimeric immunoreceptors have applications in the treatment of various cancers, particularly redirecting T cell antigen specificity via the autocrine / paracrine cytokine system utilized by human malignancies.

[0441] In certain embodiments, the chimeric cytokine receptor comprises an immunosuppressive cytokine or a cytokine receptor-binding variant thereof, a linker, a transmembrane domain, and an intracellular signaling domain.

[0442] In certain embodiments, the cytokine or cytokine receptor-binding variant thereof is selected from the group consisting of interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), and interleukin-13 (IL-13).

[0443] In certain embodiments, the linker comprises a CH2CH3 domain, a hinge domain, etc. In one embodiment, the linker comprises the CH2 and CH3 domains of IgG1, IgG4, or IgD. In one embodiment, the linker comprises a CD8α or CD4 hinge domain.

[0444] In certain embodiments, the transmembrane domain is selected from the group consisting of the alpha or beta chain of a T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PD-1.

[0445] In certain embodiments, the intracellular signaling domain is selected from the group consisting of ITAMs containing a major signaling domain and / or a costimulatory domain.

[0446] In specific embodiments, the intracellular signaling domain is selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0447] In certain embodiments, the intracellular signaling domain is selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70.

[0448] In one embodiment, the chimeric cytokine receptor comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3ζ major signaling domain.

[0449] F. Polypeptides A variety of polypeptides are contemplated herein, including, but not limited to, homing endonuclease variants, megaTALs, and fusion polypeptides. In a preferred embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NOS: 1-19 and 38. The terms "polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably according to their conventional meaning, i.e., as a sequence of amino acids, unless otherwise specified. In one embodiment, "polypeptide" includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides are not limited to a particular length; for example, they can include full-length protein sequences, fragments of full-length proteins, or fusion proteins, and can include post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, and other modifications, both naturally occurring and non-naturally occurring, known in the art.

[0450] As used herein, "isolated protein," "isolated peptide," or "isolated polypeptide," and similar terms, refer to the in vitro synthesis, isolation, and / or purification of a peptide or polypeptide molecule from its cellular environment and from association with other components of a cell, i.e., it is not significantly associated with in vivo substances.

[0451] Illustrative examples of polypeptides contemplated in certain embodiments include, but are not limited to, homing endonuclease variants, megaTALs, BiTEs, cytokines, chemokines, cytotoxins, and cytokine receptors, flip receptors, immunosuppressive signal dampers, CARs, DARICs, TCRs, and zetakines.

[0452] Polypeptides include "polypeptide variants." Polypeptide variants can differ from native polypeptides in one or more amino acid substitutions, deletions, additions, and / or insertions. Such variants can be naturally occurring or synthetically produced, for example, by modifying one or more amino acids of the above-described polypeptide sequences. For example, in certain embodiments, it may be desirable to improve the biological properties of a homing endonuclease, such as megaTAL, that binds to and cleaves a target site in the human CBLB gene by introducing one or more substitutions, deletions, additions, and / or insertions into the polypeptide. In certain embodiments, polypeptides include those having at least about 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to any of the reference sequences contemplated herein, typically where the variant retains at least one biological activity of the reference sequence.

[0453] Polypeptide variants include biologically active "polypeptide fragments." Illustrative examples of biologically active polypeptide fragments include DNA-binding domains, nuclease domains, and the like. As used herein, a "biologically active fragment" or "minimal biologically active fragment" refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the activity of a naturally occurring polypeptide. In preferred embodiments, the biological activity is binding affinity and / or cleavage activity for a target sequence. In certain embodiments, a polypeptide fragment may comprise an amino acid chain of at least 5 to about 1700 amino acids in length. In certain embodiments, the fragment comprises at least 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, 50, 55, 60, It will be appreciated that the polypeptide may be 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids in length. In certain embodiments, the polypeptide comprises a biologically active fragment of a homing endonuclease variant. In certain embodiments, the polypeptides described herein may contain one or more amino acids designated as "X." "X," when present in an amino acid SEQ ID NO:, refers to any amino acid. One or more "X" residues may be present at the N-terminus and C-terminus of the amino acid sequences set forth in the SEQ ID NOs specifically contemplated herein. If the "X" amino acid is not present, the remaining amino acid sequence set forth in the SEQ ID NO: is considered a biologically active fragment.

[0454] In certain embodiments, the polypeptide comprises a biologically active fragment of a homing endonuclease variant, e.g., SEQ ID NOs: 3-12, or megaTAL (SEQ ID NOs: 13-19). The biologically active fragment may comprise an N-terminal truncation and / or a C-terminal truncation. In certain embodiments, the biologically active fragment lacks or comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence, more preferably a deletion of 4 N-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence. In certain embodiments, the biologically active fragment lacks or comprises a deletion of 1, 2, 3, 4, or 5 C-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence, more preferably a deletion of 2 C-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence. In particularly preferred embodiments, the biologically active fragment lacks or comprises a deletion of 4 N-terminal amino acids and 2 C-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence.

[0455] In certain embodiments, an I-OnuI variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E, and / or a deletion of 1, 2, 3, 4, or 5 of the following C-terminal amino acids: R, G, S, F, V.

[0456] In certain embodiments, an I-OnuI variant comprises deletions or substitutions of 1, 2, 3, 4, 5, 6, 7, or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E, and / or deletions or substitutions of 1, 2, 3, 4, or 5 of the following C-terminal amino acids: R, G, S, F, V.

[0457] As mentioned above, polypeptides can be modified in various ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al., (1987, Methods in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, JD et al., (Molecular See Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and references cited therein. Guidance for appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0458] In certain embodiments, a variant will contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted with another amino acid having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic properties of the polypeptide to remain substantially unchanged. Modifications can be made in the polynucleotide and polypeptide structures contemplated in certain embodiments, including polypeptides containing functional molecules that encode variant or derivative polypeptides having at least about the same sequence and still possessing desirable properties. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent or even improved variant polypeptide, one skilled in the art can, for example, change one or more of the codons in the encoding DNA sequence, e.g., according to Table 1. [Table 1]

[0459] Guidance for determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR, GCG, DNA Strider, Geneious, MacVector, or Vector NTI software. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one of a family of amino acids related in their side chains. Naturally occurring amino acids are generally classified into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. In peptides or proteins, suitable conservative substitutions of amino acids are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of polypeptides do not substantially alter biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p.224).

[0460] In one embodiment, when expression of more than one polypeptide is desired, the polynucleotide sequences encoding them may be separated by an IRES sequence as disclosed elsewhere herein.

[0461] In certain embodiments, contemplated polypeptides include fusion polypeptides. In certain embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides that have at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptide segments.

[0462] In another embodiment, two or more polypeptides may be expressed as a fusion protein comprising one or more self-cleaving polypeptide sequences disclosed elsewhere herein.

[0463] In one embodiment, a fusion protein contemplated herein comprises one or more DNA binding domains, one or more nucleases, and one or more linker and / or self-cleaving polypeptides.

[0464] In one embodiment, a fusion protein contemplated herein comprises a nuclease variant; a linker or a self-cleaving peptide; and an end-processing enzyme, including but not limited to, a 5'-3' exonuclease, a 5'-3' alkaline exonuclease, and a 3'-5' exonuclease (e.g., Trex2).

[0465] Fusion polypeptides may contain one or more polypeptide domains or segments, including, but not limited to, signal peptides, cell-penetrating peptide domains (CPPs), DNA-binding domains, nuclease domains, etc., epitope tags (e.g., maltose-binding protein ("MBP"), glutathione S-transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptide linkers, and polypeptide cleavage signals. Fusion polypeptides can be linked C-terminally to C-terminally, N-terminally to N-terminally, or N-terminally to C-terminally, but are typically linked C-terminally to N-terminally. In certain embodiments, the polypeptides of the fusion protein can be in any order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides can be produced by chemical synthesis methods or by chemical conjugation between two moieties, or generally can be prepared using other standard techniques. The ligated DNA sequence comprising the fusion polypeptide is operably linked to suitable transcriptional or translational control elements, as disclosed elsewhere herein.

[0466] Fusion polypeptides optionally contain a linker that can be used to link one or more polypeptides or domains within a polypeptide. A peptide linker sequence can be used to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structure so that the polypeptide domains can perform their desired functions. Such peptide linker sequences are incorporated into fusion polypeptides using standard techniques in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) the ability to adopt a flexible, extended conformation, (2) the inability to adopt secondary structures that can interact with functional epitopes on the first and second polypeptides, and (3) the lack of hydrophobic or charged residues that can react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn, and Ser residues. Other similar neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Amino acid sequences that can be usefully used as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. Linker sequences are not required when certain fusion polypeptide segments contain non-essential N-terminal amino acid regions that can be used to separate functional domains and prevent steric interference. Preferred linkers are typically flexible amino acid subsequences synthesized as part of the recombinant fusion protein. Linker polypeptides can be 1 to 200 amino acids long, 1 to 100 amino acids long, or 1 to 50 amino acids long, including all integer values ​​therebetween.

[0467] An exemplary linker is the following amino acid sequence: glycine polymer (G) n ; glycine-serine polymer (G 1-5 S 1-5 ) n(wherein n is an integer of at least 1, 2, 3, 4, or 5); glycine-alanine polymer; alanine-serine polymer; GGG (SEQ ID NO: 39); DGGGS (SEQ ID NO: 40); TGEKP (SEQ ID NO: 41) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 42) (Pomerantz et al. 1995, supra); (GGGGS) n (wherein n=1, 2, 3, 4, or 5 (SEQ ID NO: 43) (Kim et al., PNAS 93, 1156-1160 (1996); EGKSSGSGSESKVD (SEQ ID NO: 44) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSSEQLAQFRSLD (SEQ ID NO: 45) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 46); LQRDGERP (SEQ ID NO: 47); LRQKDGGGSERP (SEQ ID NO: 48); LRQKD(GGGS)2ERP (SEQ ID NO: 49). Alternatively, flexible linkers can be modeled using computer programs capable of modeling both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994), or can be rationally designed by phage display methods.

[0468] The fusion polypeptide may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein or between the endogenous open reading frame and the polypeptide encoded by the donor repair template.In addition, the polypeptide cleavage site can be included in any linker peptide sequence.Representative polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004.Traffic,5(8);616-26).

[0469] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Representative protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, biovirus RNA-2 encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungrospherical virus) 3C-like protease, PYVF (parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to their high cleavage stringency, TEV (tobacco etch virus) protease cleavage sites are preferred in one embodiment, e.g., EXXYXQ(G / S) (SEQ ID NO: 50), e.g., ENLYFQG (SEQ ID NO: 51) and ENLYFQS (SEQ ID NO: 52) (wherein X represents any amino acid) (cleavage by TEV occurs between Q and G or Q and S).

[0470] In certain embodiments, the self-cleaving polypeptide site comprises a 2A or 2A-like site, sequence, or domain (Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041). In certain embodiments, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0471] In one embodiment, the viral 2A peptide is selected from the group consisting of a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a zowsea signa virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0472] Illustrative examples of 2A sites are provided in Table 2. [Table 2]

[0473] G. Polynucleotides In certain embodiments, polynucleotides are provided that encode one or more of the homing endonuclease variants, megaTALs, endo-processing enzymes, and fusion polypeptides contemplated herein, or one or more biologically active fragments or variants. As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides can be single-stranded or double-stranded, and can be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, genomic RNA (gRNA), positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), tracrRNA, crRNA, single guide RNA (sgRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. In certain embodiments, the polynucleotide is a polynucleotide fragment encoding one or more biologically active fragments or variants. A polynucleotide refers to a length of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10,000, or at least 15,000 or more nucleotides, as well as all intermediate lengths of nucleotides, polymeric forms of either ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. In this context, it will be readily understood that "intermediate length" means any length between the recited values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc.In certain embodiments, a polynucleotide or variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference sequence.

[0474] In certain embodiments, a polynucleotide may be codon-optimized. As used herein, the term "codon-optimized" refers to substituting codons in a polynucleotide encoding a polypeptide to increase the expression, stability, and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to, one or more of: (i) variation in codon bias between two or more organisms or genes or synthetically constructed bias tables; (ii) variation in the degree of codon bias among organisms, genes, or sets of genes; (iii) systematic variation of codons with context; (iv) variation of codons according to their decoding tRNA; (v) variation of codons according to GC % either throughout the triplet or at a single position; (vi) variation in the degree of similarity to a reference sequence, e.g., a naturally occurring sequence; (vii) variation in codon frequency cutoffs; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence upon which the design of the codon substitution set is based; (x) systematic variation of the codon set for each amino acid; and / or (xi) isolated removal of spurious translation initiation sites.

[0475] As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotide is understood to include natural bases and a wide variety of art-recognized modified bases. Such bases are generally located at the 1' position of the nucleotide sugar moiety. Nucleotides generally comprise a base, a sugar, and a phosphate group. In ribonucleic acid (RNA), the sugar is ribose; in deoxyribonucleic acid (DNA), the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group present in ribose. Representative natural nitrogenous bases include purines, adenosine (A) and guanidine (G), as well as pyrimidines, cytidine (C), and thymidine (T) (or, in the context of RNA, uracil (U)). The C-1 atom of deoxyribose is linked to the N-1 of a pyrimidine or the N-9 of a purine. Nucleotides are usually mono-, di-, or triphosphates. Nucleotide can be unmodified or modified at sugar, phosphate and / or base moiety (also referred to interchangeably as nucleotide analogue, nucleotide derivative, modified nucleotide, non-natural nucleotide and non-standard nucleotide, for example, see WO92 / 07065 and WO93 / 15187).Examples of modified nucleobases are summarized by Limbach et al., (1994, Nucleic Acids Res.22,2183-2196).

[0476] Nucleotides may also be recognized as phosphate esters of nucleosides, with esterification occurring on the hydroxyl group attached to C-5 of the sugar. As used herein, the term "nucleoside" refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are also recognized in the art to include natural bases and well-known modified bases. Such bases are generally located at the 1' position of the nucleoside sugar moiety. Nucleosides generally comprise a base and a sugar group. Nucleosides may be unmodified or modified in the sugar and / or base moieties (interchangeably referred to as nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides, or non-standard nucleosides). As also mentioned above, examples of modified nucleobases are summarized by Limbach et al., (1994, Nucleic Acids Res. 22, 2183-2196).

[0477] Illustrative examples of polynucleotides include, but are not limited to, polynucleotides encoding SEQ ID NOs: 1-19 and 38, and the polynucleotide sequences set forth in SEQ ID NOs: 20-37.

[0478] In various exemplary embodiments, polynucleotides contemplated herein include, but are not limited to, polynucleotides encoding homing endonuclease variants, megaTALs, endoprocessing enzymes, fusion polypeptides, and expression vectors, viral vectors, and transfer plasmids comprising the polynucleotides contemplated herein.

[0479] As used herein, terms such as " polynucleotide variant " and " variant " refer to a polynucleotide that shows substantial sequence identity with a reference polynucleotide sequence or polynucleotide that hybridizes with a reference sequence under strict conditions as defined below.These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution or modification of at least one nucleotide.Therefore, the terms " polynucleotide variant " and " variant " include the polynucleotide that one or more nucleotides are added or deleted, or modified, or replaced with different nucleotides.In this regard, it is well understood in the art that certain modifications, including mutation, addition, deletion and substitution, can be made to a reference polynucleotide, and thereby the modified polynucleotide maintains the biological function or activity of the reference polynucleotide.

[0480] Polynucleotide variants include polynucleotide fragments encoding biologically active fragments or variants. As used herein, the term "polynucleotide fragment" refers to at least 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 2 "A" refers to a polynucleotide fragment having a length of 7, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more nucleotides. A polynucleotide fragment refers to a polynucleotide that encodes a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution of one or more amino acids of a naturally occurring or recombinantly produced polypeptide.

[0481] In one embodiment, the polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions. Hybridization under "stringent conditions" describes a hybridization protocol in which nucleotide sequences that are at least 60% identical to each other remain hybridized. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Generally, the target sequence is present in excess, so at Tm, 50% of the probes are occupied at equilibrium.

[0482] As used herein, " sequence identity " or statements including, for example, "50% sequence identity to" refer to the degree to which sequences are identical on a nucleotide or amino acid basis in a comparison window.Therefore, "sequence identity percentage" can be calculated by comparing two optimally aligned sequences in a comparison window, determining the number of positions where identical nucleic acid bases (for example, A, T, C, G, I) or identical amino acid residues (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occur in both sequences, obtaining the number of matched positions, and dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100, thereby obtaining the percentage of sequence identity. Typically, polypeptide variants include nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein, provided that the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0483] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percent sequence identity," and "substantial identity." A "reference sequence" is at least 12, often 15-18, and often at least 25 monomer units in length, including nucleotides and amino acid residues. Because two polynucleotides can each contain (1) sequences that are similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence) and (2) sequences that are diverse between the two polynucleotides, sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least six contiguous positions, typically about 50 to about 100, more typically about 100 to about 150, over which a sequence is compared to a reference sequence of the same number of contiguous positions after optimal alignment of the two sequences. For optimal alignment of two sequences, the comparison window can contain about 20% or less of addition or deletion (i.e., gap) compared to the reference sequence (not including addition or deletion).The optimal alignment of sequences for aligning the comparison window can be performed by computer implementation of algorithms (such as GAP, BESTFIT, FASTA, and TFASTA in DNASTAR, GCG, DNA Strider, Geneious, MacVector, or Vector NTI software), or by inspection and the best alignment (i.e., the one that produces the highest percentage of homology in the comparison window) produced by any of the various methods selected.For example, the BLAST family of programs, such as those disclosed by Altschul et al., 1997, Nucl.Acids Res.25:3389, can also be referenced.A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994-1998, Chapter 15, Unit 19.3.

[0484] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that flank it in its naturally occurring state, e.g., a DNA fragment that has been removed from sequences that normally flank the fragment. In certain embodiments, an "isolated polynucleotide" refers to a complementary DNA (cDNA), recombinant polynucleotide, synthetic polynucleotide, or other polynucleotide that is not found in nature and has been created by the hand of man.

[0485] In various embodiments, the polynucleotide comprises an mRNA encoding a polypeptide contemplated herein, including but not limited to a homing endonuclease variant, a megaTAL, and an endo-processing enzyme. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0486] As used herein, the term "5' cap" or "5' cap structure" or "5' cap moiety" refers to a chemical modification incorporated at the 5' end of an mRNA. The 5' cap is involved in nuclear export, mRNA stability, and translation.

[0487] In certain embodiments, mRNAs contemplated herein contain a 5' cap comprising a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule, which 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue.

[0488] Illustrative examples of 5' caps suitable for use in certain embodiments of the mRNA polynucleotides contemplated herein include unmethylated 5' cap analogs, e.g., G(5')ppp(5')G, G(5')ppp(5')C, G(5')ppp(5')A; methylated 5' cap analogs, e.g., m 7 G(5´)ppp(5´)G、m 7 G(5´)ppp(5´)C, and m 7 G(5')ppp(5')A; dimethylated 5' cap analogs, e.g., m 2,7 G(5´)ppp(5´)G、m 2,7 G(5´)ppp(5´)C, and m 2,7 G(5')ppp(5')A; trimethylated 5' cap analogs, e.g., m 2,2,7 G(5´)ppp(5´)G、m 2,2,7 G(5´)ppp(5´)C, and m 2,2,7 G(5')ppp(5')A; dimethylated symmetric 5' cap analogues, e.g., m 7 G(5´)pppm 7 (5´)G、m 7 G(5´)pppm 7 (5´)C, and m 7 G(5´)pppm 7 (5')A; and anti-reverse 5' cap analogs, such as anti-reverse cap analogs (ARCA) caps, designated 3'O-Me-m 7 G(5´)ppp(5´)G, 2´O-Me-m 7 G(5´)ppp(5´)G, 2´O-Me-m 7 G(5´)ppp(5´)C, 2´O-Me-m 7 G(5´)ppp(5´)A、m 7 2´d(5´)ppp(5´)G、m 7 2´d(5´)ppp(5´)C、m 7 2´d(5´)ppp(5´)A, 3´O-Me-m 7 G(5´)ppp(5´)C, 3´O-Me-m 7 G(5´)ppp(5´)A、m 7 3´d(5´)ppp(5´)G、m 7 3´d(5´)ppp(5´)C、m 73'd(5')ppp(5')A, and their tetraphosphate derivatives) (see, e.g., Jemielity et al., RNA, 9:1108-1122 (2003)).

[0489] In certain embodiments, the mRNA is linked to the 5' end of the first transcribed nucleotide via a triphosphate bridge to form the m 7 7-methylguanylate ("m"), which gives G(5')ppp(5')N, where N is any nucleoside. 7 Contains a 5' cap that is 5' G').

[0490] In some embodiments, the mRNA comprises a 5' cap, and the cap is a Cap0 structure (the Cap0 structure lacks a 2'-O-methyl residue on the ribose attached to bases 1 and 2), a Cap1 structure (the Cap1 structure has a 2'-O-methyl residue at base 2), or a Cap2 structure (the Cap2 structure has a 2'-O-methyl residue attached to both bases 2 and 3).

[0491] In one embodiment, the mRNA is 7 Contains G(5´)ppp(5´)G cap.

[0492] In one embodiment, the mRNA includes an ARCA cap.

[0493] In certain embodiments, the mRNA contemplated herein comprises one or more modified nucleosides.

[0494] In one embodiment, the mRNA is selected from the group consisting of pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine , 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- Pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine Zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-Diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wyobutosine, 7-deaza-guanosine, 7-deaza and one or more modified nucleosides selected from the group consisting of 8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0495] In one embodiment, the mRNA is selected from the group consisting of pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, The nucleoside comprises one or more modified nucleosides selected from the group consisting of 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

[0496] In one embodiment, the mRNA is selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio- The nucleoside comprises one or more modified nucleosides selected from the group consisting of 1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0497] In one embodiment, the mRNA is selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6-(cis-hydroxyisopentene)-2-aminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenylade ... and one or more modified nucleosides selected from the group consisting of N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0498] In one embodiment, the mRNA comprises one or more modified nucleosides selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0499] In one embodiment, the mRNA comprises one or more pseudouridines, one or more 5-methyl-cytosines, and / or one or more 5-methyl-cytidines.

[0500] In one embodiment, the mRNA comprises one or more pseudouridines.

[0501] In one embodiment, the mRNA includes one or more 5-methyl-cytidines.

[0502] In one embodiment, the mRNA includes one or more 5-methyl-cytosines.

[0503] In certain embodiments, mRNAs contemplated herein comprise a poly(A) tail to help protect the mRNA from exonuclease degradation, stabilize the mRNA, and facilitate translation. In certain embodiments, the mRNA comprises a 3' poly(A) tail structure.

[0504] In certain embodiments, the length of the poly(A) tail is at least about 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or at least about 500 or more adenine nucleotides, or any intervening number of adenine nucleotides. 58, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 202, 203, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239 , 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, or 275 or more adenine nucleotides.

[0505] In certain embodiments, the length of the poly(A) tail is from about 10 to about 500 adenine nucleotides, from about 50 to about 500 adenine nucleotides, from about 100 to about 500 adenine nucleotides, from about 150 to about 500 adenine nucleotides, from about 200 to about 500 adenine nucleotides, from about 250 to about 500 adenine nucleotides, from about 300 to about 500 adenine nucleotides, from about 50 to about 450 adenine nucleotides, from about 50 to about 400 adenine nucleotides, from about 50 to about 350 adenine nucleotides, from about 100 to about 500 adenine nucleotides, from about 100 to about 450 adenine nucleotides, from about 100 to about 400 adenine nucleotides, from about 100 to about 350 adenine nucleotides, from about 100 to about 300 adenine nucleotides, from about 150 to about 500 adenine nucleotides, from about 150 to about 500 adenine nucleotides, The adenine nucleotides are about 450 adenine nucleotides, about 150 to about 400 adenine nucleotides, about 150 to about 350 adenine nucleotides, about 150 to about 300 adenine nucleotides, about 150 to about 250 adenine nucleotides, about 150 to about 200 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 200 to about 450 adenine nucleotides, about 200 to about 400 adenine nucleotides, about 200 to about 350 adenine nucleotides, about 200 to about 300 adenine nucleotides, about 250 to about 500 adenine nucleotides, about 250 to about 450 adenine nucleotides, about 250 to about 400 adenine nucleotides, about 250 to about 350 adenine nucleotides, or about 250 to about 300 adenine nucleotides, or any intervening range of adenine nucleotides.

[0506] Terms describing the orientation of a polynucleotide include 5' (usually the end of a polynucleotide having a free phosphate group) and 3' (usually the end of a polynucleotide having a free hydroxyl (OH) group). Polynucleotide sequences may be noted in the 5' to 3' direction or the 3' to 5' direction. With respect to DNA and mRNA, the 5' to 3' strand is called the "sense," "plus," or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA instead of thymine (T) in DNA]. With respect to DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is called the "template," "antisense," "minus," or "non-coding" strand. As used herein, the term "reverse orientation" refers to a 5' to 3' sequence written in the 3' to 5' direction or a 3' to 5' sequence written in the 5' to 3' direction.

[0507] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the DNA sequence 5'AGTC The complementary strand of ATG 3' is 3'TCAGTAC 5'. This latter sequence is often written as its reverse complement, 5'CATGACT 3', with the 5' terminus on the left and the 3' terminus on the right. A sequence equivalent to its reverse complement is called a palindrome. Complementarity can be "partial," in which only some of the nucleic acids' bases match according to the base-pairing rules. Alternatively, there can be "perfect" or "complete" complementarity between nucleic acids.

[0508] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a genetic sequence within a vector capable of expressing RNA and subsequently expressing a polypeptide. In one embodiment, the nucleic acid cassette contains a gene(s) of interest, e.g., a polynucleotide(s) of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, an enhancer, a poly(A) sequence, and a gene(s) of interest, e.g., a polynucleotide(s) of interest. A vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cassettes. The nucleic acid cassettes are oriented within the vector in a position and arrangement such that the nucleic acid within the cassette can be transcribed into RNA, translated into a protein or polypeptide if necessary, subjected to appropriate post-translational modifications required for activity in the transformed cell, and translocated to a suitable compartment for biological activity by targeting to an appropriate intracellular compartment or secretion into an extracellular compartment. Preferably, in some embodiments, the cassette has its 3' and 5' ends adapted for ready insertion into a vector and / or genome, e.g., it has a restriction endonuclease site at each end. In a preferred embodiment, the nucleic acid cassette contains the sequence of a therapeutic gene used to treat, prevent, or ameliorate a genetic disorder. The cassette can be removed and inserted as a single unit into a plasmid or viral vector.

[0509] Polynucleotides include polynucleotide(s) of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide or fusion polypeptide, or a polypeptide that serves as a template for transcription of an inhibitory polynucleotide, as contemplated herein.

[0510] Furthermore, as contemplated herein, those skilled in the art will understand that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences or fragments of variants thereof that can encode a polypeptide. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that differ due to differences in codon usage, such as polynucleotides optimized for human and / or primate codon selection, are specifically contemplated in certain embodiments. In one embodiment, a polynucleotide comprising a specific allele sequence is provided. An allele is an endogenous polynucleotide sequence that is altered as a result of one or more mutations, such as deletion, addition, and / or substitution of nucleotides.

[0511] In certain embodiments, the polynucleotide of interest comprises a donor repair template.

[0512] In certain embodiments, the polynucleotide of interest comprises an inhibitory polynucleotide, including but not limited to, an siRNA, miRNA, shRNA, ribozyme, or another inhibitory RNA.

[0513] In one embodiment, the donor repair template containing the inhibitory RNA includes one or more regulatory sequences, such as a strong constitutive pol III promoter, e.g., a human or mouse U6 snRNA promoter, a human or mouse H1 RNA promoter, or a human tRNA-val promoter, or a strong constitutive pol II promoter, as described elsewhere herein.

[0514] Regardless of the length of the coding sequence itself, the polynucleotides contemplated in certain embodiments may be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, post-translational response elements, and polynucleotides encoding self-cleaving polypeptides, epitope tags, etc., as disclosed elsewhere herein or known in the art, thereby allowing their overall length to vary significantly. Thus, it is contemplated in certain embodiments that polynucleotide fragments of almost any length can be used, with the total length preferably being limited by the ease of preparation and the intended use of the recombinant DNA protocol.

[0515] Polynucleotides can be prepared, manipulated, expressed, and / or delivered using any of a variety of well-established techniques known in the art.To express a desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector.A desired polypeptide can also be expressed by delivering mRNA encoding the polypeptide into cells.

[0516] Illustrative examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and replaceable elements, eg, Sleeping Beauty, PiggyBac.

[0517] Additional illustrative examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses.

[0518] Illustrative examples of viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40).

[0519] Illustrative examples of expression vectors include, but are not limited to, pClneo vector (Promega) for expression in mammalian cells, pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences of the polypeptides disclosed herein can be ligated into such expression vectors for expression of the polypeptides in mammalian cells.

[0520] In certain embodiments, the vector is an episomal vector, or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that can replicate without being integrated into the host's chromosomal DNA and without being gradually lost with the division of the host cell, and also means that the vector replicates extrachromosomally or episomally.

[0521] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are those untranslated regions of the vector origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, post-transcriptional regulatory elements, polyadenylation sequences, 5' and 3' untranslated regions that interact with host cell proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters, can be used.

[0522] In certain embodiments, the polynucleotide comprises a vector, including, but not limited to, an expression vector and a viral vector. A vector may contain one or more exogenous, endogenous, or heterologous regulatory sequences, such as a promoter and / or enhancer. An "endogenous regulatory sequence" is one that is naturally linked to a given gene in the genome. An "exogenous regulatory sequence" is one that is placed in juxtaposition with a gene by genetic engineering (i.e., molecular biological techniques) so that transcription of that gene is directed by the linked enhancer / promoter. A "heterologous regulatory sequence" is an exogenous sequence from a species different from the cell being genetically engineered. A "synthetic" regulatory sequence may contain elements of another endogenous and / or exogenous sequence, and / or a sequence determined in vitro or in silico to provide optimal promoter and / or enhancer activity for a particular therapy.

[0523] As used herein, the term "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In certain embodiments, promoters operated in mammalian cells include an AT-rich region located approximately 25-30 bases upstream from the site at which transcription is initiated, and / or another sequence found 70-80 bases upstream from the start of transcription is a CNCAAT region, where N can be any nucleotide.

[0524] The term "enhancer" refers to a segment of DNA that contains a sequence that can provide enhanced transcription and, in some cases, can function independently of its orientation relative to another regulatory sequence. Enhancers can function cooperatively or additively with a promoter and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA that contains a sequence that can provide both promoter and enhancer function.

[0525] The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to the functional linkage between a nucleic acid expression control sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0526] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that permits continuous or sequential transcription of an operably linked sequence. A constitutive expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that permits expression in a wide variety of cell and tissue types, or it may be a "cell-specific," "cell type-specific," "cell lineage-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that permits expression in a limited variety of cell and tissue types, respectively.

[0527] Exemplary ubiquitous expression control sequences suitable for use in certain embodiments include the cytomegalovirus (CMV) immediate early promoter, the viral Syrian virus 40 (SV40) (e.g., early or late), the Moloney murine leukemia virus (MoMLV) LTR promoter, the Rous sarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5, and P11 promoters from vaccinia virus, the short elongation factor 1-alpha (EF1a-short) promoter, and the HIV-1 virus (HIV-2) promoter. promoter, elongation factor 1-alpha (EF1a-long) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinesin (β-KIN), human ROSA These include, but are not limited to, 26 loci (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, and the dl587rev primer binding site substitution (MND) promoter with deleted negative control regions (Challita et al., J Virol. 69(2):748-55 (1995)).

[0528] In certain embodiments, it may be desirable to use cell-, cell type-, cell lineage-, or tissue-specific expression control sequences to achieve cell-type-, cell lineage-, or tissue-specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide only in a subset of cell types, cell lineages, or tissues, or at a particular developmental stage).

[0529] As used herein, "conditional expression" can refer to any type of conditional expression, including, but not limited to, inducible expression, repressible expression, expression in cells or tissues with a particular physiological, biological, or disease state, etc. This definition is not intended to exclude cell-type or tissue-specific expression. Certain embodiments provide, for example, conditional expression of a polynucleotide of interest, where expression is controlled by subjecting a cell, tissue, organism, etc. to a treatment or condition that causes expression of the polynucleotide or that causes increased or decreased expression of a polynucleotide encoded by the polynucleotide of interest.

[0530] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid or estrogen receptors (induced by treatment with the corresponding hormones), metallothionine promoters (induced by treatment with various heavy metals), MX-1 promoters (induced by interferon), the "gene switch" mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO2002 / 088346), tetracycline-dependent regulatory systems, and the like.

[0531] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments, the polynucleotide comprises at least one (typically two) site(s) for recombination mediated by the site-specific recombinase. As used herein, the term "recombinase" or "site-specific recombinase" includes excision or recombination proteins, enzymes, cofactors, or related proteins involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), which may be wild-type proteins, or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequence or fragments thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in certain embodiments include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0532] A polynucleotide can contain one or more recombination sites for any of a wide variety of site-specific recombinases. It should be understood that the target site for the site-specific recombinase is in addition to any site(s) required for integration of the vector, e.g., a retroviral or lentiviral vector. As used herein, the terms "recombination sequence," "recombination site," or "site-specific recombination site" refer to a specific nucleic acid sequence that a recombinase recognizes and binds to.

[0533] For example, one recombination site for Cre recombinase is loxP, a 34-base pair sequence containing an 8-base pair core sequence flanked by two 13-base pair inverted repeats that serve as recombinase binding sites (Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Other exemplary loxP sites include lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), and lox71 (Albert et al., 2003). al., 1995), and lox66 (Albert et al., 1995).

[0534] Suitable recognition sites for FLP recombinase include, but are not limited to, FRT (McLeod, et al., 1996), F1, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), and FRT(RE) (Senecoff et al., 1988).

[0535] Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme λ integrase, e.g., phi-c31. φC31 SSR mediates recombination only between the heterotypic sites attB (34 bp long) and attP (39 bp long) (Groth et al., 2000). attB and attP are named for the attachment sites of phage integrase to the bacterial genome and phage genome, respectively, and both contain imperfect inverted repeats to which φC31 homodimers likely bind (Groth et al., 2000). The product sites, attL and attR, are effectively inactive against further φC31-mediated recombination (Belteki et al., 2003), thereby rendering the reaction irreversible. It has been found that insertion of attB-bearing DNA into a genomic attP site is easier to catalyze than insertion of an attP site into a genomic attB site (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, a typical strategy involves positioning an attP-bearing "docking site" at a defined locus by homologous recombination, which then partners with an incoming attB-bearing sequence for insertion.

[0536] In one embodiment, a polynucleotide contemplated herein comprises a donor repair template polynucleotide flanked by a pair of recombinase recognition sites, hi certain embodiments, the repair template polynucleotide is flanked by LoxP sites, FRT sites, or att sites.

[0537] In certain embodiments, the polynucleotides contemplated herein include one or more polynucleotides of interest that encode one or more polypeptides. In certain embodiments, to achieve efficient translation of each of the multiple polypeptides, the polynucleotide sequences may be separated by one or more IRES sequences or polynucleotide sequences that encode self-cleaving polypeptides.

[0538] As used herein, "internal ribosome entry site" or "IRES" refers to an element that facilitates direct internal ribosome entry of a cistron (protein-coding region) to an initiating codon such as ATG, thereby resulting in cap-independent translation of the gene. See, e.g., Jackson et al., 1990. Trends Biochem Sci 15(12):477-83 and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES commonly used by those skilled in the art include those described in U.S. Patent No. 6,692,736. Further examples of "IRES" known in the art include IRESs obtained from picornaviruses (Jackson et al., 1990) and IRESs obtained from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP), vascular epithelial growth factor (VEGF) (Huez et al. 1998. Mol. Cell. Biol. 18(11):6178-6190), fibroblast growth factor 2 (FGF-2), and insulin-like growth factor (IGFII), translation initiation factor eIF4G, and yeast transcription factors TFIID and HAP4, encephalomyocarditis virus (EMCV) available from Novagen (Duke et al., 1992. J. Virol. 66(3):1602-9), and the VEGF IRES (Huez et al., 1998. Mol. Cell. Biol. 18(11):6178-90). IRES have also been reported in viral genomes of Picornaviridae, Dicistroviridae, and Flaviviridae species, as well as HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0539] In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0540] In certain embodiments, the polynucleotide comprises a polynucleotide having a consensus Kozak sequence and encoding a desired polypeptide. As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that significantly promotes initial binding of mRNA to the small ribosomal subunit, increasing translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 75), where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92 and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48).

[0541] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation sequence 3' of the polynucleotide encoding the expressed polypeptide. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that directs both the termination and polyadenylation of a nascent RNA transcript by RNA polymerase II. Polyadenylation sequences promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, and thus may contribute to increased translation efficiency. Cleavage and polyadenylation are directed by poly(A) sequences in the RNA. The core poly(A) sequence of mammalian pre-mRNAs has two recognition elements flanking the cleavage-polyadenylation site. Typically, the most invariant AAUAAA hexamer is located 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements, resulting in the addition of up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is an ideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence comprises an SV40 poly(A) sequence, a bovine growth hormone poly(A) sequence (BGHpA), a rabbit β-globin poly(A) sequence (rβgpA), a variant thereof, or another suitable heterologous or endogenous poly(A) sequence known in the art.

[0542] In some embodiments, the polynucleotide or cells containing the polynucleotide utilize a suicide gene, including an inducible suicide gene, to reduce the risk of direct toxicity and / or uncontrolled proliferation. In certain embodiments, the suicide gene is not immunogenic to the host containing the polynucleotide or cells. Specific examples of suicide genes that can be used are caspase-9, caspase-8, or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).

[0543] In certain embodiments, the polynucleotide contains a gene segment that causes the genetically modified cells contemplated herein to be susceptible to negative selection in vivo. "Negative selection" refers to injected cells that can be eliminated as a result of changes in the individual's in vivo condition. A negative selectable phenotype can result from the insertion of a gene that confers sensitivity to an administered drug, such as a compound. Negative selection genes are known in the art and include, but are not limited to, the herpes simplex virus type I thymidine kinase (HSV-I TK) gene, which confers sensitivity to ganciclovir; the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase.

[0544] In some embodiments, the genetically modified cells comprise a polynucleotide that further comprises a positive marker that allows for in vitro selection of cells with a negative selectable phenotype. A positive selectable marker can be a gene that, when introduced into a host cell, expresses a dominant phenotype that allows for positive selection of cells that carry the gene. Genes of this type are known in the art and include, but are not limited to, the hygromycin B phosphotransferase gene (hph), which confers resistance to hygromycin B, the aminoglycoside phosphotransferase gene (neo or aph) from Tn5, which encodes resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multidrug resistance (MDR) gene.

[0545] In one embodiment, the positive selectable marker and the negative selectable element are linked such that loss of the negative selectable element necessarily also results in loss of the positive selectable marker. In a specific embodiment, the positive and negative selectable markers are fused such that loss of one necessarily results in loss of the other. An example of a fusion polynucleotide whose expression product is a polypeptide that confers both the desired positive and negative selection characteristics described above is the hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene results in a polypeptide that confers hygromycin B resistance for in vitro positive selection and ganciclovir sensitivity for in vivo negative selection. See also PCT US91 / 08442 and PCT / US94 / 05601 publications by S.D.Lupton, which describe the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker.

[0546] Preferred positive selectable markers are derived from genes selected from the group consisting of hph, nco, and gpt, and preferred negative selectable markers are derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT, and gpt. Exemplary bifunctional selectable fusion genes contemplated in certain embodiments include, but are not limited to, genes in which the positive selectable marker is derived from hph or neo and the negative selectable marker is derived from a cytosine deaminase or TK gene or selectable marker.

[0547] In certain embodiments, polynucleotides encoding one or more nuclease variants, megaTALs, endo-processing enzymes, or fusion polypeptides can be introduced into hematopoietic cells, such as T cells, by both non-viral and viral methods. In certain embodiments, delivery of one or more polynucleotides encoding nucleases and / or donor repair templates can be provided by the same method or by different methods, and / or by the same vector or different vectors.

[0548] The term "vector" is used herein to refer to a nucleic acid molecule that can transfer or transport another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. The vector may contain a sequence that directs autonomous replication in the cell, or may contain a sequence sufficient to allow integration into host cell DNA. In certain embodiments, non-viral vectors are used to deliver one or more polynucleotides contemplated herein to T cells.

[0549] Illustrative examples of non-viral vectors include, but are not limited to, plasmids (eg, DNA or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0550] Exemplary methods of non-viral delivery of polynucleotides contemplated in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistic bombardment, virosomes, liposomes, immunoliposomes, nanoparticles, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0551] Illustrative examples of polynucleotide delivery systems suitable for use in certain contemplated embodiments include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides have been described in the literature. See, for example, Liu et al. (2003) Gene Therapy. 10:180-187 and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. Antibody-targeted, bacterially derived, non-viable nanocell delivery is also contemplated in certain embodiments.

[0552] In certain embodiments, viral vectors containing contemplated polynucleotides can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial injection) or local application, as described below. Alternatively, vectors can be delivered ex vivo to cells such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirate, tissue biopsy), or to universal donor hematopoietic stem cells, followed by reimplantation of the cells into the patient.

[0553] In one embodiment, the viral vector containing nuclease variant and / or donor repair template is directly administered to organism for in vivo cell transduction.Alternatively, naked DNA can be administered.Administered by any route that is usually used to introduce molecules into blood or tissue cells for final contact, including but not limited to injection, infusion, topical application, and electroporation.Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and certain compositions can be administered by more than one route, but certain routes can often provide more immediate and effective responses than other routes.

[0554] Illustrative examples of viral vector systems suitable for use in certain embodiments contemplated herein include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0555] In various embodiments, one or more polynucleotides encoding a nuclease variant and / or donor repair template are introduced into hematopoietic cells, e.g., T cells, by transducing the cells with a recombinant adeno-associated virus (rAAV) containing the one or more polynucleotides.

[0556] AAV is a small (approximately 26 nm), replication-deficient, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can integrate its genome into the genome of host cells. Recombinant AAV (rAAV) typically consists, at a minimum, of a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequences are approximately 145 bp in length. In a specific embodiment, rAAV comprises ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0557] In some embodiments, a chimeric rAAV is used, in which the ITR sequences are isolated from one AAV serotype and the capsid sequence is isolated from a different AAV serotype. For example, an rAAV having an ITR sequence from AAV2 and a capsid sequence from AAV6 is referred to as AAV2 / AAV6. In certain embodiments, an rAAV vector can include an ITR from AAV2 and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV includes an ITR sequence from AAV2 and a capsid sequence from AAV...

Claims

[Claim 1] The method described in the specification.

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