PDCD-1 homing endonuclease variant

Abstract

To provide improved nuclease variants, compositions, and methods of using the same for editing the human program cell death 1 (PDCD-1) gene.SOLUTION: The present disclosure generally relates to compositions comprising homing endonuclease variants and megaTALs having improved stability and activity for cleaving a target site of the human PDCD-1 gene and methods of using the same. The present disclosure also contemplates a polypeptide comprising an engineered homing endonuclease modified to improve stability, binding, and cleavage of a target site. The polypeptide includes an I-OnuI homing endonuclease (HE) variant.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 777,471, filed December 10, 2018, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Statement The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is BLBD_109_01WO_ST25.txt. The text file is 95 KB, was created on November 26, 2019, and was submitted electronically via EFS-Web concurrently with the filing of the specification.

[0003] background The present disclosure relates to genome editing compositions with improved stability and activity. More specifically, the present disclosure relates to improved nuclease variants, compositions, and methods of using the same for editing the human programmed cell death 1 (PDCD-1) gene. [Background technology]

[0004] 2. Description of Related Art The global burden of cancer doubled between 1975 and 2000. Cancer is the second leading cause of morbidity and mortality, 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 pelvic cancer, endometrial cancer, leukemia, and pancreatic cancer. The number of new cancer cases is projected to increase to 22 million within the next 20 years.

[0005] The immune system plays a critical role in detecting and combating 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). Cancer can therefore be viewed as an immunological disorder, a failure of the immune system to mount the antitumor responses necessary to sustainably suppress and eliminate disease. To more effectively combat cancer, specific immunotherapeutic interventions developed over the past few decades have focused specifically on enhancing T cell immunity. However, these treatments have yielded only isolated cases of disease remission and have had virtually no overall success. More recent therapies using monoclonal antibodies targeting molecules that inhibit T cell activation, such as CTLA-4 or PDCD-1, have shown more substantial antitumor effects; however, these treatments are also associated with substantial toxicity due to systemic immune activation.

[0006] More recently, adoptive cellular immunotherapy strategies based on the isolation, modification, expansion, and reinfusion of T cells have been explored and tested in early-stage clinical trials. T cells have often been the effector cells of choice for cancer immunotherapy due to their selective recognition and potent effector mechanisms. Although these treatments have shown mixed success rates, a minority of patients have experienced durable remissions, highlighting the as-yet-unrealized potential of T cell-based immunotherapy.

[0007] Normal recognition of tumor cell-associated antigens by cytolytic T cells initiates targeted tumor lysis and supports 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. Engineered T cell receptors (TCRs) and chimeric antigen receptors (CARs) potentially increase the applicability of T cell-based immunotherapy to many cancers and other immune disorders.

[0008] Furthermore, state-of-the-art engineered T cells remain regulated by a complex immunosuppressive tumor microenvironment composed of cancer cells, inflammatory cells, stromal cells, and cytokines. Among these components, cancer cells, inflammatory cells, and inhibitory cytokines regulate T cell phenotype and function. Collectively, the tumor microenvironment drives the terminal differentiation of T cells into exhausted T cells.

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

[0010] Programmed cell death 1 (PDCD-1) is expressed on T cells and mediates immunosuppression by binding to immunosuppressive factors present in the tumor microenvironment, such as PD-L1 and PD-L2. PD-L1 and PD-L2 expression correlates with prognosis in some human malignancies. The PD-L1 / PDCD-1 signaling pathway is one of the key regulatory pathways for T cell exhaustion. PD-L1 is abundantly expressed on cancer cells and stromal cells, and blockade of PD-L1 / PDCD-1 using monoclonal antibodies enhances T cell antitumor function. PD-L2 also binds to PDCD-1 and negatively regulates T cell function. Summary of the Invention [Means for solving the problem]

[0011] The present disclosure relates generally, in part, to compositions comprising homing endonuclease variants and megaTALs with improved stability and activity that cleave target sites in the human PDCD-1 gene, and methods of using the same.

[0012] In various embodiments, the present disclosure contemplates polypeptides that include, in part, engineered homing endonucleases that have been engineered to improve target site stability and binding and cleavage.

[0013] In various embodiments, the polypeptide comprises an I-OnuI homing endonuclease (HE) variant.

[0014] In various embodiments, an I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human programmed cell death 1 (PDCD-1) gene is provided, and the I-OnuI HE variant has the following amino acid substitutions in the I-OnuI HE amino acid sequence set forth in any one of SEQ ID NOS: 1-5, or a biologically active fragment thereof: I14T, L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70L, S72N, N75H, A76Y, K80V, T82Y, R83A, L138M, T143 N, N153V, K156R, S159P, F168G, E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R, D236Q, V238R, T240E, V261M, and G300R. In a preferred embodiment, the I-OnuI HE variant does not comprise the amino acid sequence set forth in any one of SEQ ID NOs: 15-20.

[0015] In some embodiments, 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.

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

[0017] In certain embodiments, 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, or 5 C-terminal amino acids compared to the corresponding wild-type HE.

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

[0020] In some embodiments, the biologically active fragment lacks the two C-terminal amino acids being compared.

[0021] In certain embodiments, the I-OnuI HE variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.

[0022] In certain embodiments, the I-OnuI HE variant comprises an amino acid sequence that is at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.

[0023] In certain embodiments, the I-OnuI HE variant comprises an amino acid sequence that is at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.

[0024] In certain embodiments, the I-OnuI HE variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.

[0025] In certain embodiments, the I-OnuI HE variant comprises an amino acid sequence that is at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.

[0026] In certain embodiments, the I-OnuI HE variant comprises the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.

[0027] In a specific embodiment, the I-OnuI HE variant binds to the polynucleotide sequence set forth in SEQ ID NO:8.

[0028] In a further embodiment, the I-OnuI HE variant binds to the polynucleotide sequence set forth in SEQ ID NO:10.

[0029] In a further embodiment, the polypeptide further comprises a DNA binding domain.

[0030] In some embodiments, the DNA binding domain is selected from the group consisting of a TALE DNA binding domain and a zinc finger DNA binding domain.

[0031] In certain embodiments, the TALE DNA binding domain comprises between about 9.5 TALE repeat units and about 15.5 TALE repeat units.

[0032] In additional embodiments, the TALE DNA binding domain binds to a polynucleotide sequence in the PDCD-1 gene.

[0033] In certain embodiments, the TALE DNA binding domain binds to the polynucleotide sequence set forth in SEQ ID NO:9.

[0034] In an additional embodiment, a polypeptide comprising an I-OnuI HE variant and a TALE DNA binding domain binds to and cleaves the polynucleotide sequence set forth in SEQ ID NO:10.

[0035] In a further embodiment, the polypeptide further comprises a peptide linker and an endo-processing enzyme or a biologically active fragment thereof.

[0036] In certain embodiments, the polypeptide further comprises a viral self-cleaving 2A peptide and an endo-processing enzyme or a biologically active fragment thereof.

[0037] In additional embodiments, the endo-processing enzyme or biologically active fragment thereof has 5' to 3' exonuclease, 5' to 3' alkaline exonuclease, 3' to 5' exonuclease, 5' flap endonuclease, helicase, or template-independent DNA polymerase activity.

[0038] In certain embodiments, the endo-processing enzyme comprises Trex2 or a biologically active fragment thereof.

[0039] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 7, or a biologically active fragment thereof.

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

[0041] In a further embodiment, the polypeptide cleaves the human PDCD-1 gene at the polynucleotide sequence set forth in SEQ ID NO:10.

[0042] In various embodiments, the present disclosure contemplates, in part, polynucleotides that encode the polypeptides contemplated herein.

[0043] In various embodiments, the present disclosure contemplates, in part, polynucleotides that encode the I-OnuI HE variants contemplated herein.

[0044] In various embodiments, the polynucleotide is mRNA.

[0045] In certain embodiments, the mRNA encoding the polypeptide contemplated herein comprises the polynucleotide sequence set forth in SEQ ID NO:11.

[0046] In certain embodiments, the mRNA encoding the polypeptide contemplated herein comprises the polynucleotide sequence set forth in SEQ ID NO:12.

[0047] In various embodiments, the polynucleotide is cDNA.

[0048] In certain embodiments, the mRNA transcribed from the cDNA comprises the sequence set forth in SEQ ID NO:11.

[0049] In certain embodiments, the mRNA transcribed from the cDNA comprises the sequence set forth in SEQ ID NO:12.

[0050] In certain embodiments, a vector is provided that comprises a polynucleotide encoding the intended polypeptide, I-OnuI HE variant, mRNA, or cDNA.

[0051] In certain embodiments, the vector is an expression vector, an episomal vector, or a viral vector.

[0052] In certain embodiments, the vector is an adeno-associated virus (AAV) vector.

[0053] In various embodiments, a cell is provided that comprises a polypeptide, I-OnuI HE variant, polynucleotide, mRNA, cDNA, or vector contemplated herein.

[0054] In certain embodiments, the cells are hematopoietic cells.

[0055] In some embodiments, the cell is an immune effector cell.

[0056] In certain embodiments, the cell is a T cell.

[0057] In certain embodiments, the cells are CD3+, CD4+, and / or CD8+ cells.

[0058] In certain embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell.

[0059] In additional embodiments, the cells are natural killer (NK) cells or natural killer T (NKT) cells.

[0060] In further embodiments, the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from a site of infection, ascites, pleural effusion, splenic tissue, or a tumor.

[0061] In various embodiments, the present disclosure contemplates, in part, a method of editing the human PDCD-1 gene in a cell, comprising introducing into the cell a polynucleotide encoding a polypeptide contemplated herein, wherein expression of the polypeptide generates a double-stranded break at a target site in the human PDCD-1 gene.

[0062] In some embodiments, the present disclosure contemplates, in part, a method of editing the human PDCD-1 gene in a cell, the method comprising introducing into the cell a polynucleotide encoding a polypeptide contemplated herein, wherein expression of the polypeptide generates a double-stranded break at a target site in the human PDCD-1 gene, and the break is repaired by non-homologous end joining (NHEJ).

[0063] In various embodiments, the present disclosure contemplates, in part, a method of editing the human PDCD-1 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 generates a double-stranded break at a target site in the human PDCD-1 gene, and wherein the donor repair template is integrated into the human PDCD-1 gene by homology-directed repair (HDR) at the site of the double-stranded break (DSB).

[0064] In a further embodiment, the cell is a hematopoietic cell.

[0065] In certain embodiments, the cell is a T cell.

[0066] In certain embodiments, the cells are CD3+, CD4+, and / or CD8+ cells.

[0067] In certain embodiments, the cell is an immune effector cell.

[0068] In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell.

[0069] In certain embodiments, the cells are natural killer (NK) cells or natural killer T (NKT) cells.

[0070] In certain embodiments, the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from a site of infection, ascites, pleural effusion, splenic tissue, or a tumor.

[0071] In certain embodiments, the polynucleotide encoding the polypeptide is mRNA.

[0072] In additional embodiments, a polynucleotide encoding a 3' to 5' exonuclease is introduced into the cell.

[0073] In some embodiments, a polynucleotide encoding Trex2 or a biologically active fragment thereof is introduced into a cell.

[0074] In a further embodiment, the donor repair template encodes a PDCD-1 gene or portion thereof that contains one or more mutations compared to the wild-type PDCD-1 gene.

[0075] In certain embodiments, the donor repair template encodes an engineered antigen receptor.

[0076] In further embodiments, the engineered antigen receptor is an αβTCR, a γδTCR, one or more components of DARIC, a chimeric antigen receptor, or a zetakine.

[0077] In certain embodiments, an I-OnuI HE variant is more thermostable than an I-Onu HE variant that has not been purified to enhance thermostability.

[0078] In certain embodiments, an I-OnuI HE variant is more thermostable than an I-Onu HE variant comprising the amino acid sequence set forth in any one of SEQ ID NOs: 15-20. [Brief explanation of the drawings]

[0079] [Figure 1-1] FIG. 1A shows the location of the target sites in the PDCD-1 gene and exons 1 (SEQ ID NOs: 57, 58, and 59) and 2 (SEQ ID NOs: 60, 61, and 62). [Figure 1-2] FIG. 1B shows the location of the target site in the PDCD-1 gene and exon 5 (SEQ ID NOs: 63, 64, and 65). [Figure 2] FIG. 2 shows that the introduction of stabilizing mutations into PDCD-1 HE variants increases the thermostability of the enzyme compared to the parent PDCD-1 HE variant. [Figure 3]FIG. 3 shows that increasing the thermostability of the PDCD-1 megaTAL significantly increases editing activity compared to its parent PDCD-1 megaTAL. DETAILED DESCRIPTION OF THE INVENTION

[0080] 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 NO: 6 is the amino acid sequence of a stabilized I-OnuI LHE variant reprogrammed to bind and cleave a target site in the human PDCD-1 gene. SEQ ID NO: 7 is the amino acid sequence of a megaTAL that binds to and cleaves a target site in the human PDCD-1 gene. SEQ ID NO: 8 is the I-OnuI LHE variant target site in the human PDCD-1 gene. SEQ ID NO: 9 is the TALE DNA binding domain target site in the human PDCD-1 gene. SEQ ID NO: 10 is the megaTAL target site in the human PDCD-1 gene. SEQ ID NO: 11 is the mRNA encoding the stabilized PDCD-1 megaTAL. SEQ ID NO: 12 is the mRNA encoding the stabilized PDCD-1 megaTAL. SEQ ID NO: 13 is the mRNA encoding the mouse Trex2 protein. SEQ ID NO: 14 is the amino acid sequence encoding mouse Trex2. SEQ ID NOs: 15 to 20 are the amino acid sequences of megaTALs that bind to and cleave target sites in the human PDCD-1 gene. SEQ ID NOs: 21 to 31 list the amino acid sequences of various linkers. SEQ ID NOs: 32 to 56 describe the amino acid sequences of the protease cleavage site and the self-cleaving polypeptide cleavage site.

[0081] In the above sequences, X, if present, refers to any amino acid or the absence of an amino acid.

[0082] A. Overview The present disclosure generally relates to improved genome editing compositions and methods for their use. Genome editing enzymes hold considerable promise for treating diseases, disorders, and conditions with genetic material. Until now, genome editing enzymes engineered to bind and cleave target sites in the genome have not been able to do so with high efficiency. While not intending to be bound by any particular theory, the inventors have found that genome editing enzyme activity unexpectedly increases when the enzyme is engineered to have higher thermostability. Furthermore, amino acid positions in genome editing enzymes that can be modified to increase thermostability and activity against one target can be conserved and used to increase the thermostability of other genome editing enzymes designed to bind and cleave other target sites in the PDCD-1 gene.

[0083] In certain embodiments, the genome-edited immune effector cells contemplated herein are made more resistant to exhaustion by eliminating, reducing, or attenuating PDCD-1 expression and / or signaling.

[0084] In various embodiments, the genome editing compositions and methods include nuclease variants with enhanced stability and activity designed to bind and cleave target sites in the human programmed cell death 1 (PDCD-1) gene. In certain embodiments, the intended nuclease variants can be used to introduce double-strand breaks into target polynucleotide sequences, which can be repaired by non-homologous end joining (NHEJ) in a polynucleotide template, e.g., a donor repair template, or by homology-directed repair (HDR), i.e., non-phase recombination, in the presence of a donor repair template. In certain embodiments, the intended nuclease variants can also be designed as nickases, which generate single-strand DNA breaks that can be repaired using the cell's base excision repair (BER) mechanism or homologous recombination in the presence of a donor repair template. NHEJ is error-prone, frequently resulting 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 generate an infinite number of modifications specified by the introduction of donor DNA containing the desired sequence at the target site, flanked on both sides by sequences that generate homology to regions flanking the target site.

[0085] In one preferred embodiment, the genome editing composition contemplated herein comprises a homing endonuclease variant or megaTAL that has been modified to increase stability and / or activity and targets the human PDCD-1 gene.

[0086] In one preferred embodiment, the genome editing composition contemplated herein comprises a homing endonuclease variant or megaTAL modified to increase stability and / or activity, and an endo-processing enzyme, e.g., Trex2.

[0087] In various embodiments, cells or populations of cells comprising homing endonuclease variants or megaTALs that have been modified to increase stability and / or activity are contemplated.

[0088] In various embodiments, DNA breaks are generated at target sites in the PDCD-1 gene in T cells, e.g., immune effector cells, and NHEJ of the ends of the cleaved genomic sequence may result in cells with little or no PDCD-1 expression; preferably, T cells lacking or substantially lacking functional PDCD-1 expression and / or signaling lack the ability to increase T cell exhaustion, for example. Without intending to be bound by any particular theory, the improved nucleases contemplated herein result in faster editing rates of PDCD-1 target sites, making T cells more resistant to immune suppression and T cell exhaustion, and therefore more persistent and therapeutically effective.

[0089] In various other embodiments, a homing endonuclease variant or megaTAL modified to increase stability and / or activity and a donor template, such as a nucleic acid encoding an engineered antigen receptor, are provided. The PDCD-1 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 encoding a chimeric antigen receptor.

[0090] In a preferred embodiment, the genome editing compositions and methods contemplated herein are used to edit the human PDCD-1 gene.

[0091] Thus, the methods and compositions contemplated herein represent a quantitative improvement over existing adoptive cell therapies.

[0092] Recombinant (i.e., 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, cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd 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, Volumes I and II (IRL Press, Oxford University Press USA, 1985); Current Protocols in Immunology (eds. 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, eds. 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, 1994) New York, 1991); Oligonucleotide Synthesis (ed. N. Gait, 1984); Nucleic Acid Hybridization (ed. B. Hames and S. Higgins, 1985); Transcription and Translation (ed. B. Hames and S. Higgins, 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 ed., 2010, Humana Press); Immobilized Cells And Enzymes (IRL Press, 1986); the treatise, Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (JH Miller and MPCalos, ed., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods. See In Cell And Molecular Biology (eds. Mayer and Walker, Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (eds. D.M. Weir and C.C. Blackwell, 1986); Roitt, Essential Immunology, 6th ed. (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (eds. Q.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, 1991); Annual Review of Immunology; and academic journal monographs such as Advances in Immunology.

[0093] B. Definition Before describing this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[0094] 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 method and material similar or equivalent to those described herein can be used in the implementation or testing of specific embodiments, preferred embodiments of compositions, methods and materials are described herein.For the purpose of this disclosure, the following terms are defined below.

[0095] 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 one or more) of the grammatical object of the article. By way of example, "an" means one element or one or more elements.

[0096] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0097] The term "and / or" should be understood to mean either one or both of the alternatives.

[0098] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to an amount, 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 amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0099] In one embodiment, a range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers 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.

[0100] As used herein, the term "substantially" refers to an amount, 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 more compared to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces about the same effect, e.g., a physiological effect, as the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0101] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to imply the inclusion of the specified step or component or group of steps or components, but not the exclusion of any other step or component or group of steps or components. "Consisting of" means including and limited to everything that follows the word "consisting of." Thus, the word "consisting of" indicates that the recited components are required or essential, and that no other components may be present. "Consisting essentially of" means including any components listed after the word, and is limited to other components that do not interfere with or participate in the activity or function specified in this disclosure for the recited components. Thus, the word "consisting essentially of" indicates that the recited components are required or essential, but that there are no other components that materially affect the activity or function of the recited components.

[0102] Throughout this specification, the use of "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 a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. Thus, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the forward recitation of a feature in an embodiment serves as a basis for excluding the feature in certain embodiments.

[0103] The term "in vitro" generally refers to activities occurring at a location outside of an organism, such as experiments or measurements conducted in an artificial environment outside of the organism, preferably with minimal alteration of natural conditions, or experiments or measurements on living tissue. In certain embodiments, "in vitro" procedures involve living cells or tissues taken from an organism and cultured or conditioned in a laboratory setting, usually under sterile conditions, typically for a few hours or up to about 24 hours, but up to 48 or 72 hours depending on the circumstances. In certain embodiments, such tissues or cells can be collected, frozen, and then thawed for ex vivo processing. Tissue culture experiments or procedures using living cells or tissues that last for several days or more are typically considered "in vitro," although in certain embodiments, the term may be used interchangeably with ex vivo.

[0104] The term "in vivo" generally refers to activities that occur inside an organism. In one embodiment, a cell's genome is manipulated, edited, or modified in vivo.

[0105] "Enhancement" or "promotion" or "increase" or "magnification" or "potentiation" generally refers to the ability of a nuclease variant to produce, induce, or cause a greater response (i.e., a physiological response) compared to the response caused by either a vehicle or a control. Measurable responses can include, among other things, increased stability, catalytic activity, binding affinity, persistence, cytolytic activity, and / or increased pro-inflammatory cytokines, as is apparent from understanding in the art and the description 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-fold or more (e.g., 500, 1000-fold) (including all integers and decimal points above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response caused by a vehicle or control.

[0106] "Decrease" or "reduction" or "decrease" or "reduction" or "attenuation" or "excision" or "inhibition" or "attenuation" generally refers to the ability of a nuclease variant intended herein to produce, induce, or cause a lesser response (i.e., a physiological response) compared to the response caused by either a vehicle or a control. Measurable responses can include reduced stability, off-target binding affinity, off-target cleavage specificity, T cell exhaustion, and the like. A "reduction" or "reduced" amount is typically a "statistically significant" amount and can include a reduction of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (including all integers and decimal points above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) compared to the response caused by a vehicle or control.

[0107] "Maintain" or "preserve" or "maintained" or "no change" or "no substantial change" or "no substantial decrease" generally refers to the ability of a nuclease variant to produce, induce, or cause a substantially similar or equivalent physiological response (i.e., downstream effect) compared to the response caused by either a vehicle or a control. An equivalent response is not significantly or measurably different from the response of the reference.

[0108] 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 that binds to DNA with higher binding affinity than background binding. A binding domain can be, for example, 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). In certain embodiments, a binding domain "specifically binds" to or associates with a target site with a 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 10 9 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 higher K a refers to those binding domains that have

[0109] Alternatively, affinity may be expressed in M ​​units (e.g., 10 -5 M~10 -13 The equilibrium dissociation constant (K d The affinity of a nuclease variant comprising one or more DNA binding domains for a DNA target site of interest in certain embodiments can be readily determined using conventional techniques, for example, yeast cell surface display or by binding association or displacement assays using labeled ligands.

[0110] 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 greater than background binding, or more.

[0111] The terms "selectively bind" or "selectively bound" or "selective binding" or "selectively target" describe the preferential binding of one molecule to a target molecule (binding on the target) in the presence of multiple off-target molecules. In certain embodiments, the HE or MegaTAL selectively binds to a DNA binding site on the target at a frequency that is about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 100-fold, or 1000-fold higher than the HE or MegaTAL binds to an off-target DNA target binding site.

[0112] "Site on target" refers to the target site sequence.

[0113] "Off-target" refers to a sequence that is similar, but not identical to, the target site sequence.

[0114] 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 exist. When referring to a polynucleotide sequence or SEQ ID NO: that references only one strand of a target site or target sequence, it is 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 strand. In a preferred embodiment, the target site is a sequence of the human PDCD-1 gene.

[0115] "Protein stability" refers to the net balance of forces that determine whether a protein is in its native folded structure or in a denatured (unfolded or extended) state. Protein unfolding, either partial or complete, can result in loss of function along with degradation by cellular machinery. Polypeptide stability can be measured in response to a variety of conditions, including, but not limited to, temperature, pressure, and osmotic concentration.

[0116] "Thermal stability" refers to the ability of a protein to fold properly in its native folded structure or to resist denaturation or unfolding upon exposure to temperature fluctuations. At temperatures that are not ideal, proteins may not fold efficiently into activity or may tend to unfold from activity. Proteins with increased thermal stability fold properly and retain activity over an increased temperature range compared to proteins with lower thermal stability.

[0117] "TM 50 " refers to the temperature at which 50% of the protein mass is unfolded. In certain embodiments, TM 50 is the temperature at which that amount of protein has 50% of its maximum activity. 50is a specific value determined by fitting multiple data points to a Boltsmann sigmoid curve. In one non-limiting example, the TM of a protein 50 TM is measured in a yeast surface display activity assay by expressing the protein on the yeast surface at approximately 25°C, distributing the yeast into multiple wells, exposing them to a range of higher temperatures, cooling the yeast, and then measuring the cleavage activity of the enzyme. As the temperature increases, more of the protein loses its activity signature, and therefore fewer protein-expressing cells show sufficient activity to measure cleavage by flow cytometry. The temperature at which 50% of the yeast display population is active compared to the non-heat-shocked population is known as TM. 50 is.

[0118] "Recombination" refers to the process of genetic information exchange between two polynucleotides, including, but not limited to, non-homologous end joining (NHEJ) and donor capture via homologous recombination. For purposes of this disclosure, "homologous recombination (HR)" refers to a specific form of such exchange that occurs, for example, during repair of a double-strand break in a cell 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 transfer of genetic information from the donor to the target. Without intending to be bound by any particular theory, such transfer may involve mismatch correction of heteroduplex DNA formed between the disrupted target and donor, and / or "synthesis-dependent strand annealing," in which the donor is used to resynthesize the genetic information that will become part of the target, and / or related processes. Such specified HR often results in an 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.

[0119] "NHEJ" or "non-homologous end joining" refers to the resolution 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 with distinct mutational consequences. The classical NHEJ pathway (cNHEJ) requires the KU / DNA-PKcs / Lig4 / XRCC4 complex, which ultimately religates with minimal processing, often leading to accurate repair of the break. Alternative NHEJ pathways (altNHEJ) are also effective in resolving dsDNA breaks, but these pathways are highly mutagenic and result in imprecise repair of breaks marked by insertions and deletions. Without intending to be bound by any particular theory, it is contemplated that modification of dsDNA breaks with end-processing enzymes such as exonucleases, e.g., Trex2, can increase the likelihood of imprecise repair.

[0120] "Cleavage" refers to the destruction of the covalent backbone of a DNA molecule. Cleavage can be initiated by various 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 separate single-strand cleavage events. DNA cleavage can result in the generation of either blunt or staggered ends. In certain embodiments, 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.

[0121] A "foreign" molecule is a molecule that is not normally present in a cell but is introduced into a cell by one or more genetic, biochemical, or other methods. Exemplary foreign molecules include, but are not limited to, small organic molecules, proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, modified derivatives of any of the above molecules, or any complex containing one or more of the above molecules. Methods for introducing foreign molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated delivery (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, biolistics, biopolymer nanoparticles, calcium phosphate co-precipitation, DEAE-dextran-mediated delivery, and viral vector-mediated delivery.

[0122] 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.

[0123] "Gene" refers to a DNA region that encodes a gene product, as well as all DNA regions that regulate the production of that gene product, whether or not such regulatory sequences are adjacent to the coding and / or transcribed sequence. Genes include, but are not limited to, promoter sequences, enhancers, silencers, insulators, border regions, terminators, polyadenylation sequences, post-transcriptional response elements, translation control sequences such as ribosome binding sites and internal ribosome entry sites, origins of replication, substrate binding sites, and locus control regions.

[0124] "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 mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins that are modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0125] As used herein, the term "genetically engineered" or "genetically modified" refers to the chromosomal or extrachromosomal addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. The genetic modification can be targeted to a specific site in the genome of the cell or can be non-targeted. In one embodiment, the genetic modification is site-specific. In one embodiment, the genetic modification is not site-specific.

[0126] As used herein, the term "genome editing" refers to the replacement, deletion, and / or introduction of genetic material at a target site in the genome of a cell, which restores, corrects, disrupts, and / or alters the expression and / or function of a gene or gene product. In certain embodiments, genome editing contemplated involves introducing one or more nuclease variants into a cell to generate a DNA lesion at or near the target site in the genome of the cell, optionally in the presence of a donor repair template.

[0127] As used herein, the term "gene therapy" refers to the introduction of excess genetic material into a cell's total genetic material to restore, correct, or alter the expression of a gene or gene product, or to express a therapeutic polypeptide. In certain embodiments, the introduction of genetic material into a cell's genome by genome editing to restore, correct, disrupt, or alter the expression of a gene or gene product, or to express a therapeutic polypeptide, is considered gene therapy.

[0128] Further definitions are set forth throughout this disclosure.

[0129] C. Nuclease Variants The nuclease variants contemplated in certain embodiments herein are modified to increase thermostability and enzymatic activity.The nuclease variants are suitable for genome editing of target sites in the PDCD-1 gene and comprise one or more DNA binding domains and one or more DNA cleavage domains (e.g., one or more endonuclease domains 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 nucleases that comprise one or more DNA binding domains and one or more DNA cleavage domains, wherein the nuclease is designed and / or modified from a parent or naturally occurring nuclease to bind and cleave double-stranded DNA target sequences in the PDCD-1 gene.

[0130] In certain embodiments, the nuclease variant binds and cleaves the target sequence "ATCC" in exon 1 of the PDCD-1 gene, preferably in SEQ ID NO: 8 in exon 1 of the PDCD-1 gene, more preferably in SEQ ID NO: 8 in exon 1 of the PDCD-1 gene.

[0131] Nuclease variants may be designed and / or modified from naturally occurring nucleases or from previous nuclease variants. In preferred embodiments, I-OnuI HE variants comprise increased thermostability and / or enzymatic activity compared to the parent I-OnuI HE variant. In certain embodiments, contemplated nuclease variants may further comprise one or more additional functional domains, such as a 5' to 3' exonuclease, a 5' to 3' alkaline exonuclease, a 3' to 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 exhibiting template-independent DNA polymerase activity.

[0132] Examples of nuclease variants that bind to and cleave target sequences in the PDCD-1 gene include, but are not limited to, homing endonuclease (meganuclease) variants and megaTALs.

[0133] 1. Homing endonuclease (meganuclease) variants In various embodiments, the homing endonuclease or meganuclease is engineered to increase its thermostability and enzymatic activity and to introduce a double-stranded break (DSB) at a target site in the PDCD-1 gene. In certain embodiments, the homing endonuclease variant introduces a double-stranded break in exon 1 of the PDCD-1 gene, preferably at SEQ ID NO: 8 in exon 1 of the PDCD-1 gene, more preferably at the sequence "ATCC" in SEQ ID NO: 8 in exon 1 of the PDCD-1 gene.

[0134] "Homing endonucleases" and "meganucleases" are used interchangeably and refer to naturally occurring homing endonucleases that recognize 12-45 base pair cleavage sites and are generally grouped into five families based on sequence and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK.

[0135] 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.

[0136] "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, where the homing endonuclease binds to, cleaves, and cleaves a DNA target sequence in the PDCD-1 gene and is designed and / or modified from a parent or naturally occurring homing endonuclease, and further modified to increase thermal stability and enzymatic activity. The homing endonuclease variant may be designed and / or modified from a naturally occurring homing endonuclease or from another homing endonuclease variant. Homing endonuclease variants contemplated in certain embodiments may further comprise one or more additional functional domains, e.g., an endo-processing enzyme domain of an endo-processing enzyme that exhibits 5' to 3' exonuclease, 5' to 3' alkaline exonuclease, 3' to 5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerase, or template-independent DNA polymerase activity.

[0137] Homing endonuclease (HE) variants do not exist in nature and can be obtained by recombinant DNA technology or random mutagenesis. HE variants may be obtained by making one or more amino acid changes in a naturally occurring HE or HE variant, for example, by mutating, substituting, adding, or deleting one or more amino acids. In certain embodiments, the HE variant contains one or more amino acid changes to the DNA recognition interface.

[0138] 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 that exhibits 5' to 3' exonuclease, 5' to 3' alkaline exonuclease, 3' to 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 a T cell that has an endo-processing enzyme that exhibits 5' to 3' exonuclease, 5' to 3' alkaline exonuclease, 3' to 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 may be introduced separately, for example, on 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 an IRES element.

[0139] The "DNA recognition interface" refers to the HE amino acid residues that interact with the nucleic acid target base as well as neighboring residues. For each HE, the DNA recognition interface comprises an extensive network of side chain-to-side chain and side chain-to-DNA contacts, most of which are necessarily unique for recognizing a particular nucleic acid target sequence. Thus, the amino acid sequence of the DNA recognition interface corresponding to a particular nucleic acid sequence varies widely and is characteristic of any natural HE or HE variant. As a non-limiting example, HE variants contemplated in certain embodiments can be derived by constructing a library of HE variants in which one or more amino acid residues located in the DNA recognition interface of a natural HE (or a previously generated HE variant) are altered. The library can be screened for target cleavage activity against each predicted PDCD-1 target site using a cleavage assay (see, e.g., Jarjour et al., 2009, Nuc. Acids Res. 37(20):6871-6880).

[0140] LAGLIDADG homing endonucleases (LHEs) are the best-studied family of homing endonucleases, are primarily encoded in archaea and in organelle DNA in green algae and fungi, and exhibit the highest overall DNA recognition specificity.

[0141] In one embodiment, the reprogrammed LHE or LHE variant is an I-OnuI variant. See, e.g., SEQ ID NO: 6.

[0142] In one embodiment, a reprogrammed I-OnuI targeting the PDCD-1 gene The LHE or I-OnuI variant was generated from naturally occurring I-OnuI or a biologically active fragment thereof (SEQ ID NOs: 1-5). In a preferred embodiment, a reprogrammed I-OnuI LHE or I-OnuI variant targeting the human PDCD-1 gene was generated from an existing I-OnuI variant. In one embodiment, a reprogrammed I-OnuI LHE was generated against the human PDCD-1 gene target site set forth in SEQ ID NO: 8.

[0143] In certain embodiments, the I-OnuI HE variant cleaves the PDCD-1 exon 1 target site and is selected from the group consisting of I14T, L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70L, S72N, N75H, A76Y, K80V, T82Y, R83A, L138M, T1 ... and G300R.

[0144] In some embodiments, the I-OnuI HE variant cleaves the PDCD-1 exon 1 target site and has the following amino acid substitutions: I14T, L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70L, S72N, N75H, A76Y, K80V, T82Y of I-OnuI (SEQ ID NOs: 1-5) or a biologically active fragment thereof. , R83A, L138M, T143N, N153V, K156R, S159P, F168G, E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R, D236Q, V238R, T240E, V261M, and G300R.

[0145] In certain embodiments, an I-OnuI LHE variant that binds to and cleaves the human PDCD-1 gene comprises an amino acid sequence that is at least 98% or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NO: 6 or a biologically active fragment thereof.

[0146] 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.

[0147] 2. MegaTAL In various embodiments, the megaTAL comprising the homing endonuclease variant is engineered to increase its thermostability and enzymatic activity and to introduce a double-strand break (DSB) at a target site in the PDCD-1 gene. In a particular embodiment, the megaTAL introduces a double-strand break in exon 1 of the PDCD-1 gene, preferably in SEQ ID NO: 10 in exon 1 of the PDCD-1 gene, more preferably in the sequence "ATCC" of SEQ ID NO: 10 in exon 1 of the PDCD-1 gene.

[0148] "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 in the PDCD-1 gene and optionally comprises one or more linkers and / or additional functional domains, such as a 5' to 3' exonuclease, a 5' to 3' alkaline exonuclease, a 3' to 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.

[0149] In certain embodiments, the megaTAL can be introduced into cells together with an endo-processing enzyme that exhibits 5' to 3' exonuclease, 5' to 3' alkaline exonuclease, 3' to 5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerase, or template-independent DNA polymerase activity. The megaTAL and 3' processing enzyme can be introduced separately, for example, on 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.

[0150] A "TALE DNA-binding domain" is the DNA-binding portion of a transcription activator-like effector (TALE or TAL effector), which mimics plant transcription activators and manipulates the plant transcriptome (see, e.g., Kay et al., 2007, Science 318:648-651). In certain embodiments, contemplated TALE DNA binding domains are engineered novel or from naturally occurring TALEs, such as AvrBs3 from bacterial spot pathogens Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonas euvesicatoria, and Xanthomonas oryzae, and brg11 and hpx17 from Ralstonia solanacearum. Examples of TALE protein 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.

[0151] In certain embodiments, a megaTAL comprises a TALE DNA-binding domain comprising 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 called 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 that constitute a repeat variable dipeptide (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 directs 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.

[0152] Examples of non-canonical RVDs suitable for use in certain megaTALs contemplated in certain embodiments are HH, KH, NH, NK, NQ, RH, RN, SS, NN, SN, KN for guanine (G) recognition; NI, KI, RI, HI, SI for adenine (A) recognition; NG, HG, KG, RG for thymine (T) recognition; RD, SD, HD, ND, KD, YG for cytosine (C) recognition; NV, HN for A or G recognition; and H for A or T or G or C recognition. * , H.A., K.A., N. * ,NA,NC,NS,RA,S * (( * ) means that the amino acid at position 13 is absent. Further 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.

[0153] 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, more preferably 9, 10, 11, 12, 13, 14, or 15 repeat units.

[0154] In certain embodiments, a megaTAL contemplated herein comprises a TALE DNA-binding domain comprising 3-30 repeat units and an additional single truncated TALE repeat unit comprising 20 amino acids located at the C-terminus of the set of TALE repeat units, i.e., an additional C-terminal half-TAL DNA-binding domain repeat unit (amino acids -20 to -1 of the C-cap disclosed above and elsewhere herein). Thus, in certain embodiments, a megaTAL contemplated herein comprises a TALE DNA-binding domain comprising 3.5-30.5 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, a 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, more preferably 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, or 15.5 repeat units.

[0155] In certain embodiments, a megaTAL comprises a TAL effector construct 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 from the same species. In other embodiments, one or more of the NTD, TALE repeat, and / or CTD domains are from different species.

[0156] 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, may 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.

[0157] 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, may 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 the C-terminal half-repeat unit, up 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 from at least about amino acids -20 to -1 of a Xanthomonas TALE protein (-20 is amino acid 1 of the C-terminal half-repeat unit of the C-terminal full repeat unit). In particular 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(s) 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 from at least about amino acids -20 to -1 of a Ralstonia TALE protein (-20 is amino acid 1 of the C-terminal half-repeat unit of the last 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 acids C-terminal to the last complete repeat of the TALE DNA binding domain of a Ralstonia TALE protein.

[0158] In certain embodiments, the megaTALs contemplated herein comprise 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 engineered to increase enzymatic stability and / or activity, and optionally an NTD polypeptide and / or a CTD polypeptide linked to each other with one or more linker polypeptides as otherwise contemplated herein. Without intending to be bound by any particular theory, it is contemplated that the megaTALs comprising the TALE DNA-binding domain and, optionally, the NTD and / or CTD polypeptides are 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 approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides away from the target sequence bound by the DNA-binding domain of the homing endonuclease variant. In this manner, the megaTALs contemplated herein increase the specificity and efficiency of genome editing.

[0159] In one embodiment, the megaTAL comprises a TALE DNA binding domain that binds to a nucleotide sequence within about 2, 3, 4, 5, or 6 nucleotides, preferably 2 or 4 nucleotides, upstream of the binding site of the homing endonuclease variant and the reprogrammed homing endonuclease.

[0160] In one embodiment, the MegaTAL comprises a homing endonuclease variant engineered to improve thermal stability and / or enzymatic activity and a TALE DNA binding domain that binds to the nucleotide sequence set forth in SEQ ID NO: 9, which is two nucleotides upstream of the nucleotide sequence bound and cleaved by the homing endonuclease variant (SEQ ID NO: 8). In a preferred embodiment, the MegaTAL target sequence is SEQ ID NO: 10.

[0161] In certain embodiments, a megaTAL as contemplated herein comprises one or more TALEs. The I-OnuI HE variants include DNA-binding repeat units and I-OnuI HE variants that include increased thermostability and / or enzymatic activity compared to the parent I-OnuI HE variant.

[0162] In certain embodiments, a megaTAL contemplated herein comprises an NTD, one or more TALE DNA-binding repeat units, a CTD, and an I-OnuI HE variant with increased thermal stability and / or enzymatic activity compared to the parent I-OnuI HE variant.

[0163] In certain embodiments, a megaTAL contemplated herein comprises an NTD, about 9.5 to about 15.5 TALE DNA-binding repeat units, and an I-OnuI HE variant with increased thermal stability and / or enzymatic activity compared to the parent I-OnuI HE variant.

[0164] In certain embodiments, megaTALs contemplated herein comprise 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 HE variant comprising increased thermal stability and / or enzymatic activity compared to the parent I-OnuI HE 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.

[0165] In certain embodiments, a megaTAL contemplated herein comprises the amino acid sequence set forth in SEQ ID NO:7.

[0166] In certain embodiments, a megaTAL contemplated herein is encoded by an mRNA sequence set forth in any one of SEQ ID NOs: 11 or 12.

[0167] In certain embodiments, the megaTAL comprises a TALE DNA binding domain and an I-OnuI LHE variant that binds to and cleaves the nucleotide sequence set forth in SEQ ID NO: 8 or 10. In certain embodiments, the megaTAL that binds to and cleaves the nucleotide sequence set forth in SEQ ID NO: 8 or 10 comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0168] 3. Endo-processing enzymes In certain embodiments, contemplated genome editing compositions and methods include editing a cellular genome using an I-OnuI HE variant and an end-processing enzyme that have increased thermostability and / or enzymatic activity compared to the parent I-OnuI HE variant. In certain embodiments, a single polynucleotide encodes the homing endonuclease variant and the end-processing enzyme, separated by a linker, a self-cleaving peptide sequence, e.g., 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 a single polypeptide fusion of the homing endonuclease variant and the end-processing enzyme, in addition to tandem copies of the end-processing enzyme separated by a self-cleaving peptide.

[0169] The term "endo-processing enzyme" refers to an enzyme that modifies exposed ends of polynucleotide chains. 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 ends 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 ends at endonuclease cleavage sites or at ends generated by shearing (e.g., by passing through a fine-gauge needle, heating, sonication, minibead tumbling, and spraying), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolysis, and other chemical or mechanical means, such as chemotherapeutic agents.

[0170] In certain embodiments, genome editing compositions and methods contemplated in certain embodiments include editing a cellular genome using an I-OnuI HE variant and an I-OnuI HE variant that comprises increased thermostability and / or enzymatic activity compared to the parent I-OnuI HE variant or a megaTAL and a DNA end-processing enzyme.

[0171] The term "DNA end-processing enzyme" refers to an enzyme that modifies exposed ends of DNA. DNA end-processing enzymes can modify blunt 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 at ends generated by other chemical or mechanical means, such as by shearing (e.g., by passing through a fine-gauge needle, heating, sonication, mini-bead tumbling, and spraying), ionizing radiation, ultraviolet radiation, 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.

[0172] Examples of DNA end-processing enzymes suitable for use in certain embodiments contemplated herein include, but are not limited to, 5' to 3' exonucleases, 5' to 3' alkaline exonucleases, 3' to 5' exonucleases, 5' flap endonucleases, helicases, phosphatases, hydrolases, and template-dependent DNA polymerases.

[0173] Additional examples of DNA end-processing enzymes suitable for use in certain embodiments contemplated herein include Trex2, Trex1, transmembrane domain-free Trex1, Apollo, Artemis, DNA2, Exo1, ExoT, ExoIII, Fen1, Fan1, MreII, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, Lambda exonuclease, Sox, vaccinia DNA polymerase, exonuclease I, exonuclease II, exonuclease III, exonuclease III, exonuclease I, exonuclease III, exonuclease I, exonuclease III, exonuclease I, exonuclease III, exonuclease I, exonuclease II ... These include, but are not limited to, nuclease 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.

[0174] In certain embodiments, the genome editing compositions and methods contemplated herein for editing a cellular genome comprise an I-OnuI HE variant or MegaTAL and a polypeptide comprising an exonuclease. The term "exonuclease" refers to an enzyme that cleaves a phosphodiester bond at the end of a polynucleotide chain via a hydrolysis reaction that cleaves the phosphodiester bond at either the 3' or 5' end.

[0175] Examples of exonucleases suitable for use in certain embodiments contemplated herein include, but are not limited to, hExoI, yeast ExoI, E. coli hTREX2, mouse TREX2, rat TREX2, hTREX1, mouse TREX1, rat TREX1, and rat TREX1.

[0176] In certain embodiments, the DNA end-processing enzyme is a 3' to 5' exonuclease, preferably Trex1 or Trex2, more preferably Trex2, even more preferably human or mouse Trex2.

[0177] D. Target site In various embodiments, the I-OnuI HE variant and MegaTAL bind to and cleave a target sequence in the programmed death receptor 1 (PDCD-1) gene.

[0178] In a preferred embodiment, the homing endonuclease variant or megaTAL breaks double-stranded DNA and introduces a DSB in the polynucleotide sequence set forth in SEQ ID NO:8 or 10.

[0179] In a preferred embodiment, the homing endonuclease variant or megaTAL introduces a DSB in exon 1 of the PDCD-1 gene, preferably in SEQ ID NO: 8 (or SEQ ID NO: 10) of exon 1 of the PDCD-1 gene, more preferably in the sequence "ATCC" of SEQ ID NO: 8 (or SEQ ID NO: 10) of exon 1 of the PDCD-1 gene.

[0180] In a preferred embodiment, the PDCD-1 gene is a human PDCD-1 gene.

[0181] E. Donor repair template Nuclease variants may be used to introduce DSBs into target sequences, which may be repaired via the homology-directed repair (HDR) mechanism in the presence of one or more donor repair templates.

[0182] In certain embodiments, a donor repair template is used to insert a sequence into a genome. In particularly preferred embodiments, a donor repair template is used to repair or modify a sequence in a genome.

[0183] In various embodiments, the donor repair template comprises one or more polynucleotides encoding an engineered antigen receptor.

[0184] In various embodiments, the donor repair template is introduced into a hematopoietic cell, e.g., a T cell, by transducing the cell with an adeno-associated virus (AAV), retrovirus, e.g., lentivirus, IDLV, herpes simplex virus, adenovirus, or vaccinia virus vector containing the donor repair template.

[0185] In certain embodiments, the donor repair template comprises one or more homology arms flanking the DSB site.

[0186] As used herein, the term "homologous arm" refers to the nucleic acid sequence in the donor repair template that is identical or nearly identical to the DNA sequence adjacent to the DNA break introduced by the nuclease at the target site.In one embodiment, the donor repair template comprises a 5' homologous arm that comprises a nucleic acid sequence that is identical or nearly identical to the DNA sequence 5' of the DNA break site.In one embodiment, the donor repair template comprises a 3' homologous arm that comprises a nucleic acid sequence that is identical or nearly identical to the DNA sequence 3' of the DNA break site.In a preferred embodiment, the donor repair template comprises a 5' homologous arm and a 3' homologous arm.The donor repair template can comprise homology to the genomic sequence immediately adjacent to the DSB site, or homology to the genomic sequence within any number of base pairs from the DSB site. In one embodiment, the donor repair template comprises a nucleic acid sequence that is homologous to a genomic sequence 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 lengths of homologous sequence.

[0187] Examples of suitable lengths of homology arms contemplated in certain embodiments may be independently selected and include, but are not limited to, homology arms of 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, 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, including all intervening lengths of the homology arms.

[0188] Additional 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.

[0189] In certain embodiments, the lengths of the 5' and 3' homology arms are independently selected from about 500 bp to about 1500 bp. In one embodiment, the 5' homology arm is about 1500 bp and the 3' homology arm is about 1000 bp. In one embodiment, the 5' homology arm is about 200 bp to about 600 bp and the 3' homology arm is about 200 bp to about 600 bp. In one embodiment, the 5' homology arm is about 200 bp and the 3' homology arm is about 200 bp. In one embodiment, the 5' homology arm is about 300 bp and the 3' homology arm is about 300 bp. In one embodiment, the 5' homology arm is about 400 bp and the 3' homology arm is about 400 bp. In one embodiment, the 5' homology arm is about 500 bp and the 3' homology arm is about 500 bp. In one embodiment, the 5' homology arm is about 600 bp and the 3' homology arm is about 600 bp.

[0190] The donor repair template may further comprise one or more polynucleotides, 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, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as otherwise contemplated herein.

[0191] In one embodiment, the donor repair template comprises a polynucleotide comprising the PDCD-1 gene or a portion thereof and is designed to introduce one or more mutations into the genomic PDCD-1 sequence such that a mutant PDCD-1 gene product is expressed. In one embodiment, the mutant PDCD-1 has reduced ligand binding and / or reduced intracellular signaling.

[0192] In various embodiments, the donor repair template comprises a 5' homology arm, an RNA polymerase II promoter, one or more polynucleotides encoding an immunocompetence enhancer, an immunosuppressive signal damper, or an engineered antigen receptor, and a 3' homology arm.

[0193] In various embodiments, the target site is modified with a donor repair template comprising a 5' homology arm, one or more polynucleotides encoding a self-cleaving viral peptide, e.g., T2A, an immunocompetence 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 controlled by the endogenous PDCD-1 promoter.

[0194] 1. Engineered antigen receptors In certain embodiments, genome-edited immune effector cells contemplated herein comprise an engineered antigen receptor. In one embodiment, T cells are engineered by introducing a DSB into one or more PDCD-1 genes in the presence of a donor repair template encoding the engineered antigen receptor.

[0195] In certain embodiments, the engineered antigen receptor is an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a dimerizer-regulated immune receptor complex (DARIC) or a component thereof, or a chimeric cytokine receptor.

[0196] a. Engineered TCR In certain embodiments, the genome-edited immune effector cells contemplated herein comprise engineered TCRs. In one embodiment, T cells are engineered by introducing DSBs into one or more PDCD-1 genes in the presence of a donor repair template encoding the engineered TCR. In certain embodiments, the engineered TCR is inserted into a DSB within a single PDCD-1 gene.

[0197] Naturally occurring T cell receptors contain two subunits, alpha and beta chain subunits (αβTCR) or gamma and delta chain subunits (γδTCR), each of which is a unique protein produced by recombination events in the genome of each T cell. Libraries of TCRs may be screened for their selectivity for specific target antigens. In this way, natural TCRs with high avidity and reactivity to the target antigen can be selected, cloned, and then introduced into a T cell population to be used for adoptive immunotherapy. In one embodiment, the TCR is an αβTCR. In one embodiment, the TCR is a γδTCR.

[0198] In one embodiment, T cells are engineered by introducing a donor repair template comprising a polynucleotide encoding a subunit of a TCR at a DSB in one or more PDCD-1 genes, where the TCR subunit has the ability to form a TCR that confers specificity to the T cell for 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 the ability to home to the target cell, and participates in immunologically relevant cytokine signaling. The engineered TCR also preferably binds target cells displaying the relevant tumor-associated peptide with high avidity and, optionally, mediates efficient killing of target cells presenting the relevant peptide in vivo.

[0199] The nucleic acid encoding the engineered TCR is preferably isolated from its natural context in the (naturally occurring) chromosome of the T cell and may be incorporated into a suitable vector as described elsewhere herein. In certain embodiments, both the nucleic acid and the vector containing it can be transferred to a cell, preferably a T cell. The modified T cell can then express one or more chains of the TCR encoded by the transduced nucleic acid or nucleic acid. In a preferred embodiment, the engineered TCR is a foreign TCR because it is introduced into a T cell that does 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 immune components. In contrast to engineered TCRs, CARs are engineered to bind to target antigens in an MHC-independent manner.

[0200] The TCR may be expressed with additional polypeptides attached to the amino- or carboxyl-terminal portions of the TCR, so long as the additional polypeptides do not interfere with the ability of the chain to form a functional T cell receptor and MHC-dependent antigen recognition.

[0201] Antigens recognized by engineered TCRs contemplated in certain embodiments include, but are not limited to, cancer antigens, including antigens of both hematological cancers and solid tumors. Exemplary antigens include alpha folate receptor (FRα), α vβ6 integrin, B-cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), C-type lecithin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), fibroblast activation protein ( FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), EGFR family including ErbB2 (HER2), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen progenitor gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid;These include, but are not limited to, placenta-specific 1 (PLAC1), antigen preferentially expressed in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, X-breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1);

[0202] b. Chimeric Antigen Receptor (CAR) In certain embodiments, engineered immune effector cells contemplated herein comprise one or more chimeric antigen receptors (CARs). In one embodiment, T cells are engineered by introducing a DSB into one or more PDCD-1 genes in the presence of a donor repair template encoding the CAR. In certain embodiments, the CAR is inserted into a DSB in a single PDCD-1 gene.

[0203] In various embodiments, the genome-edited T cells express a CAR that directs cytotoxicity against tumor cells. A CAR is a molecule that combines antibody-based specificity for a target antigen (e.g., a tumor antigen) with a T cell receptor activating intracellular domain to produce a chimeric protein that exhibits specific anti-tumor cell immune activity. As used herein, the term "chimeric" describes a protein composed of different protein or DNA segments from different sources.

[0204] 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 feature of CARs is their ability to redirect immune effector cell specificity, thereby leveraging the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors to induce proliferation, cytokine production, phagocytosis, or the production of molecules that can mediate cell death of target antigen-expressing cells in a major histocompatibility complex (MHC)-independent manner.

[0205] In certain embodiments, the CAR comprises an extracellular binding domain that specifically binds to a target polypeptide, e.g., a target antigen, expressed on tumor cells. 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 and confer the chimeric receptor, e.g., CAR or Daric, the ability to specifically bind to a target antigen of interest. The binding domain may comprise 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). The binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner of the biological molecule of interest.

[0206] In certain embodiments, the extracellular binding domain comprises an antibody or an antigen-binding fragment thereof.

[0207] "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, such as one recognized by immune cells. Antibodies include antigen-binding fragments, such as camelid Ig (camelid antibody or its VHH fragment), Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein ("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 (e.g., 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.

[0208] In one preferred embodiment, the binding domain is an scFv.

[0209] In another preferred embodiment, the binding domain is a camelid antibody.

[0210] In certain embodiments, the CAR is: FRα, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD7 9b, CD123, CD133, CD138, CD171, CEA, CLL-1, CS-1, CSPG4, CTAGE1, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, FAP, FCRL5, AchR, The antibody comprises an extracellular domain that binds to an antigen selected from the group consisting of GD2, GD3, GPC3, HER2, IL-11Rα, IL-13Rα2, kappa, LAGE-1A, lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MART1, MSLN, MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, survivin, TAG72, TEM1, TEM7R, TPBG, ULBP 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0211] In certain embodiments, the CAR comprises an extracellular binding domain, e.g., an antibody or antigen-binding fragment thereof, that binds to an antigen, wherein the antigen is an MHC-peptide complex, such as a class I MHC-peptide complex or a class II MHC-peptide complex.

[0212] In certain embodiments, a CAR comprises 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 binding domains so that the resulting polypeptide retains the same specific binding affinity for the same target molecule as an antibody comprising the same light chain variable region and heavy chain variable region. In certain embodiments, a CAR comprises one, two, three, four, or five or more linkers. In certain embodiments, the linker length is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, 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.

[0213] In certain embodiments, the binding domain of the CAR is followed by one or more "spacer domains," which refer to regions that distance the antigen-binding domain from the effector cell surface, allowing for proper cell-to-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). Spacer domains can be of natural, synthetic, semi-synthetic, or recombinant origin. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including one or more heavy chain constant regions, such as, but not limited to, 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.

[0214] In one embodiment, the spacer domain comprises the CH2 and CH3 of IgG1, IgG4, or IgD.

[0215] In one embodiment, the binding domain of a CAR is linked to one or more "hinge domains," which serve to position the antigen-binding domain away from the effector cell surface to allow for proper cell-cell contact, antigen binding, and activation. CARs generally comprise 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.

[0216] 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.

[0217] In one embodiment, the hinge is a PDCD-1 hinge or a CD152 hinge.

[0218] 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 cell membrane of the immune effector cell. The TM domain can be of natural, synthetic, semi-synthetic, or recombinant origin.

[0219] Exemplary TM domains may be derived from, i.e., comprise, at least the transmembrane region 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 PDCD-1.

[0220] 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 between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, connecting the TM domain and the intracellular signaling domain of the CAR. Glycine and serine linkers provide particularly suitable linkers.

[0221] In certain embodiments, the CAR comprises an intracellular signaling domain. "Intracellular signaling domain" refers to the portion of the CAR that is involved in transducing a message of effective CAR binding to a target antigen inside an immune effector cell to elicit effector cell function, such as activation, cytokine production, proliferation, or cytotoxic activity, including the release of cytokine factors into the CAR-bound target cell or other cellular responses elicited by antigen binding to the extracellular CAR domain.

[0222] The term "effector function" refers to a specialized function of a cell. Effector function of a T cell can be, for example, cytolytic activity or help or activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and instructs 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, such a truncated portion can be used in place of the entire domain, so long as it transduces the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0223] It is known that signals generated through TCR alone are 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 through 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, the CAR comprises an intracellular signaling domain that includes one or more "costimulatory signaling domains" and "primary signaling domains."

[0224] The primary signaling domain regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0225] Examples of ITAMs containing primary signaling domains 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 primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling domain and the costimulatory signaling domain may be tandemly linked to the carboxyl terminus of the transmembrane domain in any order.

[0226] 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.

[0227] Illustrative examples of such costimulatory molecules suitable for use in the CARs 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 CAR comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3ζ primary signaling domain.

[0228] 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 PDCD-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.

[0229] c.DARIC In certain embodiments, the engineered immune effector cells contain one or more components of DARIC. As used herein, the term "DARIC" refers to a dimerization agent-regulated multi-chain engineered antigen receptor. In one embodiment, T cells are engineered by introducing a DSB into one or more PDCD-1 genes 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 into a DSB in a single PDCD-1 gene.

[0230] Illustrative examples of DARIC constructs and components are disclosed in PCT Publication No. WO 2015 / 017214 and U.S. Patent Publication No. 20150266973, each of which is incorporated herein by reference in its entirety.

[0231] 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.

[0232] In certain embodiments, the multimerization domain is 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 from humans and other species (also referred to in literature as FRAP, RAPT1, or RAFT); yeast proteins including Tor1 and Tor2; and Candida FRAP homologs.The nucleotide sequence, cloning, and other information about these proteins are already known in the art.For example, the protein sequence accession number of human mTOR is GenBank accession number L34075.1 (Brown et al., Nature 369:756, 1994).

[0233] Examples of rapamycin analogs (rapalogs) include those described 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 significantly 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 either clinically or in a 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.

[0234] Other examples of rapalogs include, but are not limited to, everolimus, novolimus, pimecrolimus, ridaforimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0235] 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 for use in the fusion proteins of the present disclosure is based on the amino acid sequence of GenBank Accession No. L34075.1 and includes the 93 amino acid sequence Ile-2021 to Lys-2113 and the T2098L mutation. In certain embodiments, the FRB domain for use in Daric contemplated has the ability to bind to a complex of an FKBP protein bound to rapamycin or a rapalog thereof. In certain embodiments, the peptide sequence of the FRB domain comprises (a) a naturally occurring peptide sequence spanning at least the indicated 93 amino acid region of human mTOR or the corresponding region of a homologous protein; (b) a variant of a naturally occurring 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, only a single amino acid, 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, given the degeneracy of the genetic code, by a DNA sequence capable of selectively hybridizing to a DNA molecule encoding a naturally occurring FRB domain.

[0236] FKBP (FK506-binding protein) is a cytoplasmic receptor for macrolides such as FK506, FFK520, and rapamycin, and is highly conserved across species lineages. FKBP is a protein or protein domain capable of binding to rapamycin or its rapalogs and further forming a ternary complex with an FRB-containing protein or fusion protein. An FKBP domain may also be referred to as a "rapamycin-binding domain." Information regarding 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 are capable of binding to rapamycin or a rapalog thereof and of participating in a ternary complex with an FRB-containing protein (which can be determined by any means, direct or indirect, to detect such binding).

[0237] Examples of FKBP domains suitable for use in DARIC contemplated in certain embodiments include, but are not limited to, naturally occurring FKBP peptide sequences, preferably those isolated from the human FKBP12 protein (GenBank Accession No. AAA58476.1) or peptide sequences isolated therefrom from another human FKBP, from mouse or other mammals, or from some other animal, yeast, or fungal FKBP; variants of naturally occurring FKBP sequences 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, just one amino acid, have been deleted, inserted, or substituted; or peptide sequences encoded by nucleic acid molecules capable of selectively hybridizing to DNA molecules encoding naturally occurring FKBPs, or by DNA sequences that, due to the degeneracy of the genetic code, selectively hybridize to DNA molecules encoding naturally occurring FKBPs.

[0238] 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.

[0239] 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.

[0240] In certain embodiments, the DARIC signaling component comprises a first transmembrane domain and the DARIC binding component comprises a second transmembrane domain or a GPI anchor. Illustrative 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 PDCD-1.

[0241] In one embodiment, the DARIC signaling component comprises one or more intracellular costimulatory signaling domains and / or primary signaling domains.

[0242] Illustrative primary signaling domains suitable for use in the DARIC signaling components contemplated in certain embodiments include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In particularly preferred embodiments, the DARIC signaling component comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling domain and the costimulatory signaling domain may be tandemly attached to the carboxyl terminus of the transmembrane domain in any order.

[0243] Examples of such costimulatory molecules suitable for use in the 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 ZAP 70. 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 primary signaling domain.

[0244] In certain embodiments, the DARIC binding component comprises a binding domain, hi one embodiment, the binding domain is an antibody or antigen-binding fragment thereof.

[0245] In certain embodiments, antibodies and antigen-binding fragments thereof suitable for use with certain DARIC-binding components include, but are not limited to, murine, camelid, chimeric, humanized, or human antibodies. In preferred embodiments, the antibody or antigen-binding fragment thereof is derived from a monoclonal antibody.

[0246] Examples of antibodies and antigen-binding fragments thereof suitable for use in specific DARIC binding components include, but are not limited to, camel Ig, llama Ig, alpaca Ig, alpaca Ig, Ig NAR, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single-chain Fv proteins ("scFv"), bis-scFv, (scFv)2, minibodies, bispecific antibodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv"), and single domain antibodies (sdAb, camelid VHH, nanobodies).

[0247] In a preferred embodiment, the binding domain comprises an scFv.

[0248] In a preferred embodiment, the binding domain comprises one or more camelid VHH antibodies.

[0249] In certain embodiments, the DARIC binding component is FRα, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD13 8, CD171, CEA, CLL-1, CS-1, CSPG4, CTAGE1, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, IL-11Rα, IL-13Rα2, kappa The antibody comprises an extracellular domain that binds to an antigen selected from the group consisting of IL-1, LAGE-1A, lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MART1, MSLN, MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, survivin, TAG72, TEM1, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0250] In certain embodiments, a DARIC component contemplated herein comprises a linker or spacer connecting two proteins, polypeptides, peptides, domains, regions, or motifs.

[0251] In certain embodiments, DARIC components contemplated herein contain one or more "hinge domains," which serve to position the domains to allow proper cell-cell contact, antigen binding, and activation. In certain embodiments, the hinge is a CD8α hinge or a CD4 hinge.

[0252] In one embodiment, the 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 primary signaling domain, the binding polypeptide comprises an scFv that binds to CD19, a second multimerization domain of FKBP12, and a CD4 transmembrane domain, and the cross-linking agent is AP21967.

[0253] In one embodiment, the DARIC comprises a signaling polypeptide comprising a first multimerization domain of FRB, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ primary signaling domain, the binding polypeptide comprises an scFv that binds to CD19, a second multimerization domain of FKBP12, and a CD4 transmembrane domain, and the cross-linking agent is rapamycin, temsirolimus, or everolimus.

[0254] d. Zetakine In certain embodiments, the engineered immune effector cells contemplated herein comprise one or more chimeric cytokine receptors. In one embodiment, T cells are engineered by introducing a DSB into one or more PDCD-1 genes in the presence of a donor repair template encoding a CAR. In certain embodiments, the chimeric cytokine receptor is inserted into the DSB of a single PDCD-1 gene.

[0255] In various embodiments, genome-edited T cells express chimeric cytokine receptors that direct cytotoxicity toward tumor cells. Zetakines are chimeric transmembrane immunoreceptors that include an extracellular domain containing a soluble receptor ligand linked to a cell surface, transmembrane domain, and a support domain that can connect the extracellular domain to an intracellular signaling domain. When expressed on the surface of T lymphocytes, zetakines direct T cell activity toward those cells that express the receptor for which the soluble receptor ligand is specific. Zetakine chimeric immunoreceptors are particularly applicable to the treatment of various cancers, redirecting the antigen specificity of T cells via the autocrine / paracrine cytokine system utilized by human malignancies.

[0256] 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.

[0257] 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).

[0258] 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.

[0259] In certain embodiments, the transmembrane domain is selected from the group consisting 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 PDCD-1.

[0260] In certain embodiments, the intracellular signaling domain is selected from the group consisting of ITAMs containing a primary signaling domain and / or a costimulatory domain.

[0261] 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.

[0262] 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.

[0263] 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 the CD3ζ primary signaling domain.

[0264] F. Polypeptides Various polypeptides are contemplated herein, including, but not limited to, homing endonuclease variants and megaTALs engineered to increase thermostability and / or enzymatic activity, as well as fusion polypeptides. In a preferred embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NOs: 6 and 7. The terms "polypeptide," "peptide," and "protein" are used interchangeably in their conventional sense, i.e., according to the amino acid sequence, 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 may include full-length protein sequences, fragments of full-length proteins, or fusion proteins, and may include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and other modifications, both naturally occurring and non-naturally occurring, known in the art.

[0265] As used herein, "isolated protein," "isolated peptide," or "isolated polypeptide," etc., refers 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., not significantly associated with substances in vivo. In certain embodiments, the isolated polypeptide is a synthetic polypeptide, a semi-synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0266] Polypeptides include "polypeptide variants." Polypeptide variants may differ from naturally occurring polypeptides in one or more amino acid substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or synthetically produced, e.g., by modifying one or more amino acids of the polypeptide sequence. 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 PDCD-1 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, and typically the variants retain at least one biological activity of the reference sequence.

[0267] In a preferred embodiment, the polypeptide variant comprises a homing endonuclease or megaTAL engineered to increase its thermostability and / or activity. The I-OnuI HE polypeptide or a fragment thereof can be reprogrammed to bind to and cleave a target site. In certain embodiments, the reprogrammed I-OnuI HE variant has relatively lower thermostability and / or activity compared to the parent I-OnuI HE. In a preferred embodiment, the I-OnuI homing endonuclease or a fragment thereof is engineered to bind to and cleave a target site and increase the thermostability and / or activity of the enzyme.

[0268] Polypeptide variants include biologically active "polypeptide fragments." Examples of biologically active polypeptide fragments include DNA-binding domains, nuclease domains, and the like. As used herein, the term "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, the 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 , 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 sequence set forth in a particular SEQ ID NO contemplated herein. If the "X" amino acid is not present, the remaining amino acid sequence set forth in the SEQ ID NO may be considered a biologically active fragment.

[0269] In certain embodiments, the polypeptide comprises a biologically active fragment of a homing endonuclease variant, e.g., SEQ ID NO: 6, or MegaTAL (SEQ ID NO: 7). 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, and more preferably comprises 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, and more preferably comprises a deletion of 2 C-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence. In certain 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.

[0270] 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.

[0271] In certain embodiments, an I-OnuI variant comprises a deletion or substitution 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 or substitution of 1, 2, 3, 4, or 5 of the following C-terminal amino acids: R, G, S, F, V.

[0272] 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 or 2 of the following C-terminal amino acids: F, V.

[0273] In certain embodiments, an I-OnuI variant comprises a deletion or substitution 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 or substitution of 1 or 2 of the following C-terminal amino acids: F, V.

[0274] As mentioned above, polypeptides may be modified in various ways, including amino acid substitution, deletion, truncation, and insertion. Such manipulation methods 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 changes 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 Biology of the Gene, 4th ed., Benjamin / Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance regarding 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).

[0275] In certain embodiments, variants contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid with similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydrophobic properties of the polypeptide to remain substantially unchanged. Modifications may be made in the polynucleotide and polypeptide structures contemplated in certain embodiments, including polypeptides that have at least approximately the functional molecule encoding a variant or derivative polypeptide with desired characteristics, yet still obtain a polypeptide. If it is desired to modify the amino acid sequence of a polypeptide to generate an equivalent or improved variant polypeptide, one skilled in the art can, for example, modify one or more codons in the encoding DNA sequence, for example, according to Table 1.

[0276] [Table 1] Table 1 - Amino acid codons

[0277] Guidance for determining which amino acid residues may be substituted, inserted, or deleted in a particular embodiment without abolishing biological activity can be found in DNASTAR, DNA Conservative amino acid changes can be found using computer programs well known in the art, such as Strider, Geneious, MacVector, or Vector NTI software. Conservative amino acid changes involve substituting one of a family of amino acids related in its side chain. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. Suitable conservative substitutions of amino acids in peptides or proteins are known to those of skill in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0278] In one embodiment where expression of more than one polypeptide is desired, the polynucleotide sequences encoding them may be separated by an IRES sequence, as disclosed elsewhere herein.

[0279] In certain embodiments, intended polypeptides include fusion polypeptides. In certain embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptide segments.

[0280] In another embodiment, two or more polypeptides may be expressed as a fusion protein containing one or more self-cleaving polypeptide sequences, as disclosed elsewhere herein.

[0281] In one embodiment, a fusion protein contemplated herein comprises one or more DNA binding domains and one or more nucleases, and one or more linker and / or self-cleaving polypeptides.

[0282] In one embodiment, a fusion protein contemplated herein comprises a nuclease variant; a linker or self-cleaving peptide; and an end-processing enzyme, including, but not limited to, a 5' to 3' exonuclease, a 5' to 3' alkaline exonuclease, and a 3' to 5' exonuclease (e.g., Trex2).

[0283] Fusion polypeptides may contain one or more polypeptide domains or segments, including, but not limited to, a signal peptide, a cell-penetrating peptide domain (CPP), a DNA-binding domain, a nuclease domain, etc., an epitope tag (e.g., moltose-binding protein ("MBP"), glutathione S-transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), a polypeptide linker, and a polypeptide cleavage signal. Fusion polypeptides are typically joined C-terminally to N-terminally, but they may also be joined C-terminally to C-terminally, N-terminally to N-terminally, or N-terminally to C-terminally. In certain embodiments, the polypeptides of the fusion protein may be in any order. Fusion polypeptides or fusion proteins may also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is retained. Fusion polypeptides may be produced by chemical synthesis methods or by chemical conjugation between two moieties, or generally may 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.

[0284] Fusion polypeptides may optionally contain a linker that can be used to link one or more polypeptides or domains within the polypeptide. A peptide linker sequence can be utilized 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 accommodate a flexible, extended conformation, (2) the ability to accommodate a secondary structure that can interact with functional epitopes on the first and second polypeptides, and (3) the absence of hydrophobic or charged residues that can react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn, and Ser residues. Other near-neutral amino acids, such as Thr and Ala, can also be used in linker sequences. Amino acid sequences that can be usefully employed 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 if a particular fusion polypeptide segment contains a non-essential N-terminal amino acid region 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 in length, 1 to 100 amino acids in length, or 1 to 50 amino acids in length, including all integer values ​​in between.

[0285] Exemplary linkers include the following amino acid sequences: glycine polymer (G) n ; glycine-serine polymer (G 1-5 S 1-5 ) n(wherein n is at least 1, 2, 3, 4, or 5); glycine-alanine polymers; alanine-serine polymers; GGG (SEQ ID NO: 21); DGGGS (SEQ ID NO: 22); TGEKP (SEQ ID NO: 23) (e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 24) (Pomerantz et al., 1995, supra); (GGGGS) n (wherein n=1, 2, 3, 4, or 5) (SEQ ID NO: 25) (Kim et al., PNAS 93, 1156-1160 (1996); EGKSSGSGSESKVD (SEQ ID NO: 26) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 27) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 28); LQRDGERP (SEQ ID NO: 29); LRQKDGGGSERP (SEQ ID NO: 30); LRQKD(GGGS)2ERP (SEQ ID NO: 31). Alternatively, flexible linkers can be modeled using computer programs capable of modeling both the DNA binding site and the peptide itself (Desjarlais and Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994)) or by phage display methods.

[0286] 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. Furthermore, a polypeptide cleavage site can be inserted into any linker peptide sequence. Typical 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).

[0287] 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). Exemplary 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, biovirus 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 Tungro Spherical Virus) 3C-like protease, PYVF (Parsnip Yellow Fleck 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: 32), e.g., ENLYFQG (SEQ ID NO: 33) and ENLYFQS (SEQ ID NO: 34), where X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S).

[0288] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving peptide or a ribosomal skipping sequence.

[0289] Examples of ribosomal skipping sequences include, but are not limited to, 2A or 2A-like sites, sequences, or domains (see 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.

[0290] In one embodiment, the viral 2A peptide is selected from the group consisting of a foot-and-mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a zosea asigna virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a telirovirus 2A peptide, and an encephalopathy virus 2A peptide.

[0291] Examples of 2A sites are provided in Table 2.

[0292] [Table 2] Table 2: Exemplary 2A sites include the following sequences:

[0293] In a preferred embodiment, the polypeptide comprises an I-OnuI HE variant or megaTAL that binds to and cleaves a target site in the PDCD-1 gene and further comprises increased thermostability and / or enzymatic activity compared to the parent enzyme.

[0294] G. Polynucleotides In certain embodiments, polynucleotides encoding one or more homing endonuclease variants and megaTALs engineered to increase thermostability and / or enzymatic activity, as contemplated herein, and fusion polypeptides are provided. As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded and may 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), ribozymes, 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. Polynucleotide refers to a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides, 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 10000, or at least 15000 or more nucleotides in length, or modified forms of either type of nucleotide, as well as all intermediate lengths. It is readily understood that "intermediate length" in this context 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.

[0295] In certain embodiments, polynucleotides may be codon-optimized. As used herein, the term "codon optimization" 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 within an organism, gene, or set of genes; (iii) systematic variation of codons with context; (iv) variation of codons with their decoding tRNAs; (v) variation of codons with GC % either overall or at any single position in triplicates; (vi) variation in the degree of similarity to a reference sequence, e.g., a naturally occurring sequence; (vii) variation in codon frequency cutoff; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence on 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 start sites.

[0296] As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are understood to include natural bases and a wide variety of art-recognized modified bases. Such bases are typically 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, while in deoxyribonucleic acid (DNA), the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group present in ribose. Typical natural nitrogenous bases include purines, adenosine (A) and guanidine (G), and 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 monophosphate, diphosphate, or triphosphate. Nucleotides can be unmodified or modified at the sugar, phosphate, and / or base moieties (also referred to interchangeably as nucleotide analogs, nucleotide derivatives, modified nucleotides, non-natural nucleotides, and non-standard nucleotides; see, e.g., WO 92 / 07065 and WO 93 / 15187). Examples of modified nucleobases are reviewed by Limbach et al. (1994, Nucleic Acids Res. 22, 2183-2196).

[0297] Nucleotides may also be considered as phosphate esters of nucleosides, with esterification occurring at 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 recognized in the art to include natural bases and also include 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 can be unmodified or modified in the sugar and / or base moieties (also referred to interchangeably as nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides, or non-standard nucleosides). Also as noted above, examples of modified nucleobases are described in Limbach et al. (1994, Nucleic Acids Res. 22, 2183-2196).

[0298] Exemplary polynucleotides include, but are not limited to, polynucleotides encoding SEQ ID NOs:6 and 7, and the polynucleotide sequences set forth in SEQ ID NOs:11 and 12.

[0299] In various exemplary embodiments, polynucleotides contemplated herein include, but are not limited to, polynucleotides encoding homing endonuclease variants, megaTALs, endo-processing enzymes, fusion polypeptides, and expression vectors, viral vectors, and transfer plasmids comprising the polynucleotides contemplated herein.

[0300] As used herein, the terms "polynucleotide variant" and "variant" refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence, or hybridizes to a reference sequence under stringent 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. Thus, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides have been added or deleted, or modified, or replaced with different nucleotides. In this regard, it is well known in the art that certain changes, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, thereby allowing the modified polynucleotide to retain the biological function or activity of the reference polynucleotide.

[0301] 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) of 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. The target sequence is generally present in excess, so that at Tm, 50% of the probes are occupied at equilibrium.

[0302] The "sequence identity" listed, or for example, "50% identical sequence" used herein, refers to the degree to which sequences are identical on a nucleotide-to-nucleotide basis or an amino acid-to-amino acid basis over a comparison window.Therefore, "sequence identity percentage" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where the same nucleic acid base (for example, A, T, C, G, I) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences, and obtaining the number of matched positions, 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 to obtain the percentage of sequence identity. Included are 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, and typically, the polypeptide variant retains at least one biological activity of the reference polypeptide.

[0303] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" comprises nucleotides and amino acid residues that are at least 12 monomeric units in length, often 15-18 monomeric units, and often at least 25 monomeric units in length. Two polynucleotides may each contain (1) similar sequences between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence) and (2) divergent sequences 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, usually about 50 to about 100, more commonly about 100 to about 150, and a sequence is compared to the reference sequence over the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may contain no more than about 20% additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning the comparison window can be performed by computerized implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA) in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI, USA, or by inspection and best alignment (i.e., resulting in the highest homology across the comparison window) generated by any of a variety of selected methods. See, for example, the BLAST family of programs disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389. For a detailed discussion of sequence analysis, see Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994-1998, Chapter 15, Unit 19.3.

[0304] 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), a recombinant polynucleotide, a synthetic polynucleotide, or other polynucleotide that does not occur in nature and has been created by the hand of man. In certain embodiments, the isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

[0305] 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.

[0306] 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.

[0307] In certain embodiments, mRNAs contemplated herein include 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 may then be methylated to generate an N7-methyl-guanylate residue.

[0308] 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 analogs, 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 analog (ARCA) cap (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,m7 3'd(5')ppp(5')A and their tetraphosphate derivatives) (e.g., Jemielity et al., RNA, 9:1108-1122 (2003)).

[0309] In certain embodiments, the mRNA is attached to the 5' end of the first transcribed nucleotide via a triphosphate bridge, 7 7-methylguanylic acid ("m"), which gives G(5')ppp(5')N, where N is any nucleoside. 7 It contains a 5' cap that is

[0310] In some embodiments, the mRNA comprises a 5' cap, wherein the cap is a Cap0 structure (the Cap0 structure lacks 2'-O-methyl residues on the ribose attached to bases 1 and 2), a Cap1 structure (the Cap1 structure has 2'-O-methyl residues attached to both bases 2 and 3), or a Cap2 structure (the Cap2 structure has 2'-O-methyl residues).

[0311] In one embodiment, the mRNA is 7 Contains a G(5')ppp(5')G cap.

[0312] In one embodiment, the mRNA includes an ARCA cap.

[0313] In certain embodiments, the mRNA contemplated herein comprises one or more modified nucleosides.

[0314] 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, wybutosine, 7-deaza-guanosine, 7-deaza- The modified nucleoside comprises 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.

[0315] 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- It comprises one or more modified nucleosides selected from the group consisting of 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.

[0316] 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-1-methyl-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, The nucleotides include 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.

[0317] 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-isopentenyl adenosine, 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.

[0318] 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.

[0319] In one embodiment, the mRNA includes one or more pseudouridines, one or more 5-methyl-cytosines, and / or one or more 5-methyl-cytidines.

[0320] In one embodiment, the mRNA includes one or more pseudouridines.

[0321] In one embodiment, the mRNA includes one or more 5-methyl-cytidines.

[0322] In one embodiment, the mRNA includes one or more 5-methyl-cytosines.

[0323] 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.

[0324] 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. , 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, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 41, 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.

[0325] 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, adenine nucleotides, about 50 to about 400 adenine nucleotides, about 50 to about 350 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 100 to about 450 adenine nucleotides, about 100 to about 400 adenine nucleotides, about 100 to about 350 adenine nucleotides, about 100 to about 300 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 150 to about 500 adenine nucleotides 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 The amino acid sequence may be a nucleotide sequence of 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.

[0326] 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 can be annotated in the 5' to 3' direction or the 3' to 5' direction. For DNA and mRNA, the 5' to 3' strand is designated 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]. For DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is designated the "template," "antisense," "minus," or "non-coding" strand. As used herein, the term "reverse" 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.

[0327] 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'AGT The complement of CTATTG 3' is 3'TCAGTAC 5'. The latter sequence is often oriented with the 5' end to the left and the 3' end to the right, 5'ATG GACT 3' is written as a reverse complement. A sequence equivalent to its reverse complement is said to be a palindromic sequence. Complementarity can be "partial," where only a portion of the nucleic acid bases match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between the nucleic acids.

[0328] 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 a polypeptide. In one embodiment, the nucleic acid cassette contains a gene of interest, e.g., a polynucleotide 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 of interest, e.g., a polynucleotide 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 positionally and sequentially oriented within the vector so that the nucleic acids within the cassette can be transcribed into RNA, translated into a protein or polypeptide, if necessary, undergo appropriate post-translational modifications necessary for activity in transformed cells, and translocated to the appropriate compartment for biological activity by targeting for secretion to the appropriate intracellular or extracellular compartment. Preferably, the cassette has its 3' and 5' ends adapted for insertion into a vector, e.g., it has restriction endonuclease sites 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.

[0329] Polynucleotides include polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide or fusion polypeptide, or a polynucleotide that serves as a template for transcription of an inhibitory polynucleotide, as intended herein.

[0330] Furthermore, those skilled in the art will understand that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that can encode fragments of the polypeptides or variants thereof contemplated herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage, such as polynucleotides optimized for human and / or primate codon preferences, 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.

[0331] In certain embodiments, the polynucleotide of interest comprises a donor repair template.

[0332] 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.

[0333] In one embodiment, the donor repair template comprising the inhibitory RNA comprises one or more regulatory sequences, e.g., a strong constitutive pol III promoter, e.g., a human or mouse U6 snRNA promoter, a human and mouse H1 RNA promoter, or a human tRNA-val promoter, or a strong constitutive pol II promoter, e.g., as described elsewhere herein.

[0334] In certain embodiments, contemplated polynucleotides, regardless of the length of the coding sequence itself, may be combined with other DNA sequences and may include polynucleotides encoding promoters and / or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, 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-transcriptional response elements, and self-cleaving polypeptides, as well as epitope tags, as disclosed elsewhere herein or known in the art, thereby allowing for considerable variation in overall length. Thus, in certain embodiments, polynucleotide fragments of almost any length may be used, with the overall length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0335] Polynucleotides can be prepared, manipulated, expressed, and / or delivered using any of a variety of established techniques known and available in the art. To express a desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. The desired polypeptide can also be expressed by delivering mRNA encoding the polypeptide into cells.

[0336] Examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, eg, Sleeping Beauty, PiggyBac.

[0337] Additional 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.

[0338] 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 virus), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (e.g., SV40).

[0339] Exemplary 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.

[0340] 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 integration into the host chromosomal DNA and without gradual loss from dividing host cells, meaning that the vector replicates extrachromosomally or episomally.

[0341] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are untranslated regions of the vector, including, but not limited to, origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, post-transcriptional regulatory elements, polyadenylation sequences, and 5' and 3' untranslated regions, that interact with host cell proteins to effect 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 and inducible promoters, may be used.

[0342] 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.

[0343] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are commonly found downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation sequence 3' of the polynucleotide encoding the polypeptide to be expressed. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that directs both the termination and polyadenylation of nascent RNA transcripts by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thus contributing to improved 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, a nearly invariant AAUAAA hexamer is present 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 is an SV40 poly(A) sequence, bovine growth hormone poly(A) sequence (BGHpA), rabbit β-globin poly(A) sequence (rβgpA), a variant thereof, or another suitable heterologous or endogenous poly(A) sequence known in the art. In certain embodiments, the poly(A) sequence is synthetic.

[0344] In certain embodiments, polynucleotides encoding one or more nuclease variants, megaTALs, endo-processing enzymes, or fusion polypeptides may be introduced into hematopoietic cells, e.g., 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 may be effected by the same method or by different methods, and / or by the same vector or by different vectors.

[0345] The term "vector" is used herein to refer to a nucleic acid molecule that can transmit or transport another nucleic acid molecule. The transmitted nucleic acid is generally inserted, for example, into a vector nucleic acid molecule. The vector may contain a sequence that directs autonomous replication within a cell, or may contain a sequence sufficient to allow integration into host cell DNA. In certain embodiments, a non-viral vector is used to deliver one or more polynucleotides contemplated herein to T cells.

[0346] Examples of non-viral vectors include, but are not limited to, plasmids (eg, DNA or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0347] Exemplary methods of non-viral delivery of polynucleotides contemplated in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran mediated delivery, gene guns, and heat shock.

[0348] 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.

[0349] H. Compositions and Formulations In certain embodiments, contemplated compositions may include one or more homing endonuclease variants and megaTALs engineered to increase thermostability and / or enzymatic activity, polynucleotides, vectors comprising the same, and genome editing compositions and genome-edited cell compositions as contemplated herein. In certain embodiments, contemplated genome editing compositions and methods are useful for editing target sites in the human programmed cell death 1 (PDCD-1) gene in a cell or cell population. In a preferred embodiment, the genome editing composition is used to edit the PDCD-1 gene in hematopoietic cells, such as T cells or immune effector cells.

[0350] In various embodiments, compositions contemplated herein comprise an I-OnuI HE variant engineered for increased thermostability and / or enzymatic activity, and optionally an end-processing enzyme, such as a 3'-5' exonuclease (Trex2). The I-OnuI HE variant may be in the form of mRNA that is introduced into cells via the polynucleotide delivery methods disclosed above, such as electroporation, lipid nanoparticles, etc. In one embodiment, a composition comprising an mRNA encoding an I-OnuI HE variant or megaTAL, and optionally a 3'-5' exonuclease, is introduced into cells via the polynucleotide delivery methods disclosed above. The composition can be used to generate genome-edited cells or populations of genome-edited cells by error-prone NHEJ.

[0351] In various embodiments, the compositions contemplated herein include a donor repair template. The composition may be delivered to cells that express or will express an I-OnuI HE variant and, optionally, an end-processing enzyme. In one embodiment, the composition may be delivered to cells that express or will express an I-OnuI HE variant or a megaTAL and, optionally, a 3'-5' exonuclease. Expression of the gene editing enzyme in the presence of the donor repair template can be used to generate genome-edited cells or genome-edited cell populations via HDR.

[0352] In certain embodiments, the composition comprises cells containing one or more homing endonuclease variants engineered for increased thermostability and / or enzymatic activity, and a megaTAL, polynucleotide, or vector comprising the same. In certain embodiments, the cells may be autologous (autologous) or non-autologous (non-autologous, e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "autologous" refers to cells derived from the same subject. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the reference cell. As used herein, "syngeneic" refers to cells that are genetically identical to the reference cell but from a different subject. As used herein, "xenogeneic" refers to cells of a different species from the reference cell. In preferred embodiments, the cells are obtained from a mammalian subject. In more preferred embodiments, the cells are obtained from a primate subject, optionally a non-human primate. In most preferred embodiments, the cells are obtained from a human subject.

[0353] An "isolated cell" refers to a cell obtained from an in vivo tissue or organ and substantially free of extracellular matrix, that does not occur in nature, e.g., a non-naturally occurring cell, a modified cell, an engineered cell, a recombinant cell, etc.

[0354] As used herein, the term "population of cells" refers to a plurality of cells that may consist of any number and / or combination of homogeneous or heterogeneous cell types.

[0355] In a preferred embodiment, the cell or cell population is a hematopoietic cell, more preferably an immune cell, even more preferably a T cell.

[0356] The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, immune effector cells, regulatory T cells, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be T helper cells (HT1; CD4 + T cells)CD4 + T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - The T cells may be T cells or any other subset of T cells. In one embodiment, the T cells are immune effector cells. In one embodiment, the T cells are NKT cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells and memory T cells.

[0357] In various embodiments, the cell or population of cells comprises an immune effector cell. An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell-killing activity, secretion of cytokines, induction of ADCC and / or CDC). Exemplary immune effector cells contemplated in certain embodiments are T lymphocytes, particularly cytotoxic T cells (CTL; ​​CD8 + T cells), TILs, and helper T cells (HTLs; CD4 + In one embodiment, the immune effector cells comprise natural killer (NK) cells. In one embodiment, the immune effector cells comprise natural killer T (NKT) cells.

[0358] T cells can be obtained from a number of sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from a site of infection, ascites, pleural effusion, splenic tissue, and tumors.

[0359] In certain embodiments, compositions contemplated herein comprise a population of cells, an I-OnuI HE variant, and optionally, a donor repair template. In certain embodiments, compositions contemplated herein comprise a population of cells, an I-OnuI HE variant, an end-processing enzyme, and optionally, a donor repair template. The I-OnuI HE and / or end-processing enzyme may be in the form of mRNA introduced into cells via the polynucleotide delivery methods disclosed above.

[0360] In certain embodiments, compositions contemplated herein comprise a population of cells, an I-OnuI HE variant or megaTAL engineered to increase the enzyme's thermostability and / or activity, and optionally a donor repair template. In certain embodiments, compositions contemplated herein comprise a population of cells, an I-OnuI HE variant or megaTAL, a 3'-5' exonuclease, and optionally a donor repair template. The I-OnuI HE variant, megaTAL, and / or 3'-5' exonuclease may be in the form of mRNA introduced into cells via the polynucleotide delivery methods disclosed above.

[0361] In certain embodiments, the population of cells comprises genetically modified immune effector cells.

[0362]

[0363] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0364] Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings contemplated herein that certain changes and modifications can be made without departing from the spirit or scope of the appended claims. The following examples are provided for purposes of illustration only, and not for purposes of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially similar results. [Example]

[0365] Example 1 Reprogramming I-OnuI HE variants to increase thermostability PDCD-1 is expressed on the T cell membrane after antigen receptor stimulation and activation. PDCD-1 contains a signal peptide, an extracellular IgV-like domain, a transmembrane domain, and an intracellular tail containing both an immunoreceptor tyrosine-based inhibitory motif (ITIM, consensus sequence S / I / V / LxYxxI / V / L) and an immunoreceptor tyrosine-based switch motif (ITSM, consensus sequence TxYxxV / I) (Figure 1A and Figure 1B).

[0366] Using a yeast surface display assay, we identified mutations that increased the stability of I-OnuI HE. Multiple I-OnuI HEs were subjected to random mutagenesis via PCR across the entire open reading frame. These mutant libraries were expressed in yeast, and the TM of the library was analyzed. 50 The I-OnuI HE variants were then selected for active nuclease activity after heat shock. After two rounds of selection, the I-OnuI HE variants were sequenced using either PacBio or Sanger sequencing to determine the identity and frequency of mutations at each position. Mutations were then grafted into the I-OnuI HE variants targeting exon 1 of the PDCD-1 gene. The thermostability mutations significantly increased the TM of the PDCD-1 I-OnuI HE variants compared to the parent enzyme. 50 increased by 16°C (Figure 2).

[0367] The effect of the stabilizing mutations on PDCD-1 editing was measured by comparing the editing rate of a parent megaTAL lacking the stabilizing mutation (SEQ ID NO: 18) with a megaTAL containing the stabilizing mutation (SEQ ID NO: 7). MegaTAL mRNA was prepared by in vitro transcription, cotranscribed with an anti-inverted cap analog (ARCA), and enzymatically polyadenylated with poly(A) polymerase. Purified mRNA was used to measure PDCD-1 editing efficiency in primary human T cells.

[0368] Primary human peripheral blood mononuclear cells (PBMCs) from two donors were activated with anti-CD3 and anti-CD28 antibodies and cultured in the presence of 250 U / mL IL-2. Three days after activation, cells were electroporated with megaTAL mRNA. Transfected T cells were expanded for an additional 7–10 days, and editing efficiency was measured using sequencing across the PD-1 target site and Tracking of Indels by Decomposition (TIDE, see Brinkman et al., 2014) (Figure 3). Without the stabilizing mutation, the PDCD-1 megaTAL showed low levels of editing (<20%), while the stabilizing PDCD-1 megaTAL increased editing activity to nearly 80%.

[0369] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments, along with the full range of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure. The present invention provides, for example, the following items. (Item 1) 1. A polypeptide comprising an I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human programmed cell death 1 (PDCD-1) gene, wherein the I-OnuI HE variant has the following amino acid substitutions in the I-OnuI HE amino acid sequence set forth in any one of SEQ ID NOS: 1 to 5, or a biologically active fragment thereof: I14T, L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70L, S72N, N75H, A76Y, K80V, T82Y, R83A, L138M, T143N, N1 53V, K156R, S159P, F168G, E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R, D236Q, V238R, T240E, V261M, and G300R. (Item 2) 2. The polypeptide of item 1, wherein the I-OnuI HE variant comprises an amino acid sequence that is at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof. (Item 3) 3. The polypeptide of claim 1, wherein the I-OnuI HE variant comprises the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof. (Item 4) 4. The polypeptide of any one of items 1 to 3, wherein the I-OnuI HE variant binds to the polynucleotide sequence set forth in SEQ ID NO:8. (Item 5) 5. The polypeptide of any one of items 1 to 4, wherein the I-OnuI HE variant binds to the polynucleotide sequence set forth in SEQ ID NO: 10. (Item 6) Item 7. The polypeptide according to any one of Items 1 to 5, further comprising a DNA-binding domain. 7. The polypeptide of item 6, 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 8) 8. The polypeptide of item 7, wherein the TALE DNA binding domain comprises about 9.5 TALE repeat units to about 15.5 TALE repeat units. (Item 9) The polypeptide of item 7 or 8, wherein the TALE DNA binding domain binds to the polynucleotide sequence set forth in SEQ ID NO: 9. (Item 10) 10. The polypeptide of item 9, wherein the polypeptide binds to and cleaves the polynucleotide sequence set forth in SEQ ID NO: 10. (Item 11) 11. The polypeptide of any one of items 1 to 10, further comprising a peptide linker and an endo-processing enzyme or a biologically active fragment thereof. (Item 12) 11. The polypeptide of any one of items 1 to 10, further comprising a viral self-cleaving 2A peptide and an endo-processing enzyme or a biologically active fragment thereof. (Item 13) 13. The polypeptide of claim 11, wherein the endo-processing enzyme or a biologically active fragment thereof has 5' to 3' exonuclease, 5' to 3' alkaline exonuclease, 3' to 5' exonuclease, 5' flap endonuclease, helicase, or template-independent DNA polymerase activity. (Item 14) 14. The polypeptide according to any one of items 11 to 13, wherein the endo-processing enzyme comprises Trex2 or a biologically active fragment thereof. (Item 15) 15. The polypeptide of any one of items 1 to 14, wherein the polypeptide comprises an amino acid sequence that is at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 7 or a biologically active fragment thereof. (Item 16) 16. The polypeptide according to any one of items 1 to 15, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7 or a biologically active fragment thereof. (Item 17) A polynucleotide encoding the polypeptide according to any one of items 1 to 16. (Item 18) 17. An mRNA encoding the polypeptide according to any one of items 1 to 16. (Item 19) 19. The mRNA of item 18, wherein the mRNA comprises the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. (Item 20) Item 21. The mRNA according to Item 18, wherein the mRNA comprises the sequence set forth in SEQ ID NO: 11. Item 22. The mRNA according to Item 18, wherein the mRNA comprises the sequence set forth in SEQ ID NO: 12. A cDNA encoding the I-OnuI HE variant according to any one of items 1 to 16. (Item 23) 23. The cDNA of item 22, wherein the mRNA transcribed from the cDNA comprises the sequence set forth in SEQ ID NO: 11. (Item 24) 23. The cDNA of item 22, wherein the mRNA transcribed from the cDNA comprises the sequence set forth in SEQ ID NO: 12. (Item 25) A vector comprising a polynucleotide encoding the polypeptide according to any one of Items 1 to 16; a polynucleotide encoding the mRNA according to any one of Items 18 to 21; or a polynucleotide encoding the cDNA according to any one of Items 22 to 24. (Item 26) 26. The vector of item 25, wherein the vector is an expression vector, an episomal vector, or a viral vector. (Item 27) 27. The vector of item 26, wherein the vector is an adeno-associated virus (AAV) vector. (Item 28) A cell comprising the polypeptide according to any one of Items 1 to 16, the polynucleotide according to Item 17, the mRNA according to any one of Items 18 to 21, the cDNA according to any one of Items 22 to 24, or the vector according to any one of Items 25 to 27. (Item 29) 29. The cell of item 28, wherein the cell is a hematopoietic cell. (Item 30) 30. The cell of item 28 or item 29, wherein the cell is an immune effector cell. (Item 31) 31. The cell according to any one of items 28 to 30, wherein the cell is a T cell. (Item 32) The cells are CD3 + , CD4 + , and / or CD8 + 32. The cell according to any one of items 28 to 31, which is a cell. (Item 33) 33. The cell of any one of items 28 to 32, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. (Item 34) 31. The cell according to any one of items 28 to 30, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. (Item 35) 35. The cell of any one of items 28 to 34, wherein the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor. (Item 36) 36. The cell of any one of items 28 to 35, wherein the cell comprises one or more altered PDCD-1 alleles. (Item 37) 17. A method for editing the human PDCD-1 gene in a cell, the method comprising introducing into the cell a polynucleotide encoding a polypeptide according to any one of items 1 to 16, wherein expression of the polypeptide results in a double-stranded break at a target site in the human PDCD-1 gene. (Item 38) 17. A method for editing the human PDCD-1 gene in a cell, the method comprising introducing into the cell a polynucleotide encoding a polypeptide according to any one of items 1 to 16, wherein expression of the polypeptide causes a double-stranded break at a target site in the human PDCD-1 gene, and the break is repaired by non-homologous end joining (NHEJ). (Item 39) 17. A method for editing the human PDCD-1 gene in a cell, comprising introducing into the cell a polynucleotide encoding a polypeptide described in any one of items 1 to 16 and a donor repair template, wherein expression of the polypeptide generates a double-strand break at a target site in the human PDCD-1 gene, and the donor repair template is incorporated into the human PDCD-1 gene by homology-directed repair (HDR) at the site of the double-strand break (DSB). (Item 40) 40. The method according to any one of items 37 to 39, wherein the cells are hematopoietic cells. (Item 41) 41. The method of any one of items 37 to 40, wherein the cells are immune effector cells. (Item 42) 42. The method according to any one of items 37 to 41, wherein the cells are T cells. (Item 43) The cells are CD3 + , CD4 + , and / or CD8 + 43. The method according to any one of items 37 to 42, wherein the cell is a cell. (Item 44) 44. The method of any one of items 37 to 43, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. (Item 45) 42. The method of any one of items 37 to 41, wherein the cells are natural killer (NK) cells or natural killer T (NKT) cells. (Item 46) 46. ​​The method of any one of items 37 to 45, wherein the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor. (Item 47) 47. The method according to any one of items 37 to 46, wherein the polynucleotide encoding the polypeptide is mRNA. (Item 48) 48. The method according to any one of items 37 to 47, wherein a polynucleotide encoding a 3' to 5' exonuclease is introduced into the cell. (Item 49) 49. The method of any one of items 37 to 48, wherein a polynucleotide encoding Trex2 or a biologically active fragment thereof is introduced into the cell. (Item 50) 50. The method of any one of items 39 to 49, wherein the donor repair template encodes a PDCD-1 gene or a portion thereof that comprises one or more mutations compared to a wild-type PDCD-1 gene. (Item 51) 50. The method of any one of items 39 to 49, wherein the donor repair template encodes an engineered antigen receptor. (Item 52) Item 53. The method of Item 51, wherein the engineered antigen receptor is an engineered antigen receptor. 53. The method of claim 52, wherein the engineered antigen receptor is one or more components of an αβTCR, a γδTCR, a DARIC, a chimeric antigen receptor, or a zetakine. (Item 54) 54. The method of any one of items 37 to 53, wherein the I-OnuI HE variant is more thermostable than an I-Onu HE variant that has not been purified to increase its thermostability. (Item 55) 54. The method of any one of Items 37 to 53, wherein the I-OnuI HE variant is more thermostable than an I-Onu HE variant comprising an amino acid sequence set forth in any one of SEQ ID NOs: 15 to 20.

Claims

[Claim 1] The invention described in this specification.

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