Optimized engineered nucleases specific for the human t cell receptor alpha constant region gene

Abstract

The invention includes engineered nucleases that recognize and cleave a recognition sequence within the first exon of the human T cell receptor (TCR) alpha constant region gene. The engineered meganucleases can exhibit at least one optimized property, such as enhanced (i.e., increased) specificity or cleavage efficiency, as compared to the first generation meganuclease TRC 1-2x.87EE. The invention also includes methods of using such engineered nucleases to make genetically modified cells, as well as uses of such cells in pharmaceutical compositions and in methods of treating diseases such as cancer.

Description

TECHNICAL FIELD

[0001] The present invention relates to the fields of oncology, cancer immunotherapy, molecular biology, and recombinant nucleic acid technology. In particular, the present invention relates to optimized engineered nucleases specific for recognition sequences in human T cell receptor alpha constant region genes. The invention further relates to the use of such recombinant meganucleases in methods of producing genetically modified T cells and methods of using such cells to treat diseases, including cancer, in a subject.

[0002] References to sequence lists submitted as text files via EFS-WEB

[0003] This application contains a Sequence Listing which has been submitted via EFS-Web in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on April 11, 2019, is named P109070028WO00-SEQ, and is 2700 bytes in size. Background of the Invention

[0005] T cell adoptive immunotherapy is a promising approach to cancer treatment. This strategy utilizes isolated human T cells that are genetically modified to enhance their specificity for particular tumor-associated antigens. Genetic modification can involve expression of a chimeric antigen receptor or an exogenous T cell receptor to graft antigen specificity onto the T cell. In contrast to an exogenous T cell receptor, a chimeric antigen receptor derives its specificity from the variable domain of a monoclonal antibody. Thus, T cells expressing a chimeric antigen receptor (CAR T cells) induce tumor immunoreactivity in a major histocompatibility complex non-restricted manner. T cell adoptive immunotherapy has been used as a clinical therapy for a number of cancers, including B cell malignancies (e.g., acute lymphoblastic leukemia, B cell non-Hodgkin lymphoma, acute myeloid leukemia, and chronic lymphocytic leukemia), multiple myeloma, neuroblastoma, glioblastoma, advanced glioma, ovarian cancer, mesothelioma, melanoma, prostate cancer, pancreatic cancer, and the like.

[0006] Despite its potential use as a cancer treatment, adoptive immunotherapy with CAR T cells is limited, in part, by expression of endogenous T cell receptors on the cell surface. Upon administration to an allogeneic patient, CAR T cells expressing endogenous T cell receptors can recognize major and minor histocompatibility antigens, which can lead to the development of graft versus host disease (GVHD). As a result, clinical trials have focused primarily on the use of autologous CAR T cells, in which a patient's T cells are isolated, genetically modified to incorporate a chimeric antigen receptor, and then reinfused into the same patient. The autologous approach provides immunologic tolerance to the administered CAR T cells; however, this approach is limited by the time and expense required to generate patient-specific CAR T cells after a patient is diagnosed with cancer.

[0007] Accordingly, it would be advantageous to develop "off-the-shelf" CAR T cells prepared using T cells from third party, healthy donors that have reduced expression of endogenous T cell receptors and do not elicit GVHD upon administration. Such a product could be generated and validated prior to diagnosis and supplied to a patient as soon as necessary. Thus, there is a need to develop allogeneic CAR T cells that lack endogenous T cell receptors to prevent the occurrence of GVHD.

[0008] Genetic modification of genomic DNA can be performed using site-specific, rare-cleaving endonucleases that are engineered to recognize DNA sequences in a target locus. Homing endonucleases are a group of naturally occurring nucleases that recognize 15-40 base pair cleavage sites commonly found in plant and fungal genomes. They are often associated with parasitic DNA elements such as group I self-splicing introns and inteins. They naturally facilitate homologous recombination or gene insertion at specific locations in the host genome by creating double-stranded breaks in the chromosome, which recruits cellular DNA repair machinery (Stoddard (2006), Q. Rev. Biophys. 38:49-95). Homing endonucleases are generally classified into four families: the LAGLIDADG (SEQ ID NO: 2) family, the GIY-YIG family, the His-Cys box family, and the HNH family. These families are characterized by structural motifs that influence catalytic activity and recognition sequences. For example, members of the LAGLIDADG (SEQ ID NO: 2) family are characterized by having one or two copies of the conserved LAGLIDADG (SEQ ID NO: 2) motif (see Chevalier et al. (2001), Nucleic Acids Res. 29(18):3757-3774). LAGLIDADG (SEQ ID NO: 2) homing endonucleases with a single copy of the LAGLIDADG (SEQ ID NO: 2) motif form homodimers, while members with two copies of the LAGLIDADG (SEQ ID NO: 2) motif are found as monomers.

[0009] I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG (SEQ ID NO: 2) family of homing endonucleases, which recognizes and cleaves a 22 base pair recognition sequence in the chloroplast chromosome of the alga Chlamydomonas reinhardtii. Genetic selection techniques have been used to alter the wild-type I-CreI cleavage site preference (Sussman et al. (2004), J. Mol. Biol. 342:31-41; Chames et al. (2005), Nucleic Acids Res. 33:e178; Seligman et al. (2002), Nucleic Acids Res. 30:3870-9, Arnould et al. (2006), J. Mol. Biol. 355:443-58). Recently, a method for the rational design of single LAGLIDADG (SEQ ID NO: 2) homing endonucleases was described that enables the comprehensive redesign of I-CreI and other homing endonucleases to target a wide variety of DNA sites, including sites in mammalian, yeast, plant, bacterial, and viral genomes (WO 2007 / 047859).

[0010] As first described in WO 2009 / 059195, I-CreI and its engineered derivatives are typically dimeric, but can be fused as a single polypeptide using a short peptide linker that connects the C-terminus of the first subunit to the N-terminus of the second subunit (Li et al. (2009), Nucleic Acids Res. 37:1650-62; Grizot et al. (2009), Nucleic Acids Res. 37:5405-19). Thus, a functional “single-chain” meganuclease can be expressed from a single transcript.

[0011] The use of nucleases to disrupt expression of endogenous TCRs has been disclosed, including the use of small hairpin RNA, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), megaTALs, and CRISPR systems (e.g., Osborn et al. (2016), Molecular Therapy 24(3):570-581; Eyquem et al. (2017), Nature 543:113-117; U.S. Patent No. 8,956,828; U.S. Publication No. US2014 / 0301990; U.S. Publication No. US2012 / 0321667).

[0012] Specific uses of engineered meganucleases to cleave DNA targets in the human TCRa constant region gene have also been previously disclosed. For example, International Publication No. WO 2014 / 191527 discloses variants of the I-Onul meganuclease that are also engineered to target a recognition sequence within exon 1 of the TCRa constant region gene (‘527 published SEQ ID NO: 3). Although the ‘527 publication discusses that chimeric antigen receptors can be expressed in TCR knockout cells, the authors do not disclose inserting CAR-encoding sequences into the meganuclease cleavage site.

[0013] Further, in International Publication Nos. WO 2017 / 062439 and WO 2017 / 062451, Applicants disclosed engineered meganucleases that are specific for recognition sequences in exon 1 of the TCRa constant region gene. These include the “TRC 1-2 meganucleases” that are specific for the TRC 1-2 recognition sequence in exon 1 (SEQ ID NO: 5). The ‘439 and ‘451 publications also disclose methods of targeting insertion of CAR-encoding sequences or exogenous TCR-encoding sequences into the TRC 1-2 meganuclease cleavage site.

[0014] In the present invention, Applicants have made improvements to the nucleases and methods taught in the prior art. Through extensive experimentation, Applicants have generated novel second generation TRC 1-2 meganucleases that comprise unique, unpredictable combinations of residues and that are unexpectedly superior to the first generation TRC 1-2 meganucleases taught in the ‘439 and ‘451 applications. For example, the second generation TRC 1-2 meganucleases of the present invention have improved (i.e., increased) specificity and reduced off-target cleavage, exhibit reduced persistence in cells following mRNA expression, are superior functionally (e.g., enhanced / increased TCR knockout, enhanced / increased CAR knockin, enhanced / increased CAR T expansion, improved CAR T cell phenotype, etc.) when used to generate CAR T cells, and produce improved CAR T cell populations when used in comprehensive CAR T cell manufacturing processes. SUMMARY

[0015] The present invention provides engineered meganucleases that recognize and cleave a recognition sequence within the first exon of the human T cell receptor (TCR) alpha constant region gene (SEQ ID NO: 3). Such meganucleases can be used to disrupt the TCR alpha constant region gene, and thus the expression and / or function of the cell surface TCR. The meganuclease cleavage can disrupt gene function by mutagenesis through non-homologous end joining or by facilitating the introduction of an exogenous polynucleotide into the gene via homologous recombination. In some embodiments, the introduced exogenous polynucleotide comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), such that the meganuclease can be used to generate allogeneic CAR T cells that lack an endogenous TCR. In some embodiments, the presently disclosed engineered meganucleases exhibit at least one optimized feature compared to the first generation meganuclease TRC 1-2x.87EE. Such optimized features include improved (i.e., increased) specificity resulting in reduced off-target cleavage, reduced persistence in cells (e.g., from mRNA expression), and / or enhanced (i.e., increased) efficiency of modification of the TCR alpha constant region gene. In addition, cells that have been genetically modified with the presently disclosed engineered meganucleases exhibit improved properties compared to cells that have been genetically modified with the TRC 1-2x.87EE meganuclease, including reduced off-target cleavage and effects thereof, reduced persistence of the meganuclease in the cells, enhanced (i.e., increased) CAR T expression, and lower differentiation. Further, populations of cells into which the presently disclosed meganucleases (or nucleic acids encoding the same) have been introduced have a greater percentage of modified cells and a greater percentage of less differentiated cells compared to those populations of cells in which the TRC 1-2x.87EE meganuclease (or nucleic acids encoding the same) has been introduced.

[0016] The present invention further provides methods comprising delivering an engineered meganuclease protein or a gene encoding an engineered meganuclease to a eukaryotic cell to produce a genetically modified eukaryotic cell. Accordingly, further provided are genetically modified eukaryotic cells and populations thereof, as well as pharmaceutical compositions comprising the same. Also provided are methods of immunotherapy for treating cancer by administering a genetically modified T cell or population thereof, wherein the T cell expresses a receptor for a tumor-specific antigen (e.g., a CAR or an exogenous TCR).

[0017] Accordingly, in one aspect, the present application provides an engineered meganuclease that recognizes and cleaves a TRC 1-2 recognition sequence (SEQ ID NO: 5) in exon 1 of the human TCRa constant region gene (SEQ ID NO: 3). The engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and wherein the second subunit binds a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the amino acid sequence corresponding to residues 24-79 of the presently disclosed TRC 1-2L.1592 (the amino acid sequence of which is set forth in SEQ ID NO: 7), or at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the amino acid sequence corresponding to residues 24-79 of the presently disclosed TRC 1-2L.1775 meganuclease (the amino acid sequence of which is set forth in SEQ ID NO: 8).

[0018] In some embodiments, the HVR2 region comprises the amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7 or 8, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.

[0019] In some embodiments, the HVR2 region comprises residues corresponding to residues 24, 26, 42, 44, 46, 48, 50, 70, 71, 72, and 73 of SEQ ID NO: 7.

[0020] In some embodiments, the HVR2 region comprises residues corresponding to residues 24, 26, 38, 42, 46, 48, 50, and 70 of SEQ ID NO: 8.

[0021] In some embodiments, the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 7 or SEQ ID NO: 8.

[0022] In some embodiments, the HVR2 region comprises residues corresponding to residues 48, 50, 71, 72, and 73 of SEQ ID NO: 7.

[0023] In some embodiments, the HVR2 region comprises residues corresponding to residues 48 and 50 of SEQ ID NO: 8.

[0024] In some embodiments, the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 48, 50, 68, 70, 71, 72, 73, 75, and 77 of SEQ ID NO: 7 or SEQ ID NO: 8.

[0025] In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 7 or 8.

[0026] In some embodiments, the HVR2 region comprises residues 24-79 of SEQ ID NO: 7 or 8.

[0027] In particular embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to an amino acid sequence corresponding to residues 7-153 of SEQ ID NO: 7 or 8. In some embodiments, the second subunit comprises an amino acid sequence having at least 93% sequence identity to an amino acid sequence corresponding to residues 7-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises an amino acid sequence having at least 94% sequence identity to an amino acid sequence corresponding to residues 7-153 of SEQ ID NO: 8.

[0028] In some embodiments, the second subunit comprises an amino acid sequence corresponding to residues 7-153 of SEQ ID NO: 7 or 8, with at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.

[0029] In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 7 or 8.

[0030] In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 7 or 8.

[0031] In some embodiments, the second subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 7 or 8.

[0032] In some embodiments, the second subunit comprises a residue corresponding to residue 139 of SEQ ID NO: 7 or 8.

[0033] In particular embodiments, the second subunit comprises residues 7-153 of SEQ ID NO: 7 or 8.

[0034] In some such embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7 or 8. In some embodiments, the HVR1 region comprises an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7 or 8, with at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.

[0035] In some embodiments, the HVR1 region comprises residues corresponding to residues 219 and 231 of SEQ ID NO: 7.

[0036] In some embodiments, the HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 7 or 8.

[0037] In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 7 or 8.

[0038] In particular embodiments, the HVR1 region comprises residues 215-270 of SEQ ID NO: 7 or 8.

[0039] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the amino acid sequence corresponding to residues 198-344 of SEQ ID NO: 7 or 8. In some embodiments, the first subunit comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence corresponding to residues 198-344 of SEQ ID NO: 7 or 8. In particular embodiments, the first subunit comprises the amino acid sequence corresponding to residues 198-344 of SEQ ID NO: 7 or 8, with at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.

[0040] In some embodiments, the first subunit comprises G, S, or A at the residue corresponding to residue 210 of SEQ ID NO: 7 or 8.

[0041] In some embodiments, the first subunit comprises E, Q, or K at the residue corresponding to residue 271 of SEQ ID NO: 7 or 8.

[0042] In some embodiments, the first subunit comprises the residue corresponding to residue 271 of SEQ ID NO: 7 or 8.

[0043] In particular embodiments, the first subunit comprises residues 198-344 of SEQ ID NO: 7 or 8.

[0044] In some embodiments, the first subunit of the engineered meganuclease has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the amino acid sequence corresponding to residues 198-344 of SEQ ID NO: 7 or 8, and the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the amino acid sequence corresponding to residues 7-153 of SEQ ID NO: 7 or 8. In particular embodiments, the first subunit of the engineered meganuclease has at least 99% sequence identity to the amino acid sequence corresponding to residues 198-344 of SEQ ID NO: 7 or 8, and the second subunit comprises an amino acid sequence having at least 93% sequence identity to the amino acid sequence corresponding to residues 7-153 of SEQ ID NO: 7 or 8. In some embodiments, the first subunit and / or the second subunit can comprise up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions relative to residues 198-344 and residues 7-153 of SEQ ID NO: 7 and 8, respectively.

[0045] In some embodiments, the engineered meganuclease comprises a linker covalently joining the first subunit and the second subunit.

[0046] In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 7 or 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0047] In particular embodiments, the engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 7 or 8.

[0048] In some embodiments, the engineered meganuclease exhibits at least one of the following optimized features as compared to TRC 1-2x.87EE meganuclease as set forth in SEQ ID NO: 9: improved (i.e., increased) specificity, reduced intracellular persistence, and increased (i.e., improved) efficiency of modification of human TCRa constant region genes.

[0049] In particular embodiments, the engineered meganuclease that recognizes and cleaves within a human TCRa constant region gene a recognition sequence comprising SEQ ID NO: 5 comprises a first subunit and a second subunit, wherein the first subunit comprises: (a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity to residues 198-344 of SEQ ID NO: 7 or 8; and (b) an HVR1 region having at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7 or 8; wherein the second subunit comprises: (a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity to residues 7-153 of SEQ ID NO: 7 or 8; and (b) an HVR2 region having at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7 or 8.

[0050] In particular embodiments, the engineered meganuclease recognizing and cleaving a recognition sequence within a human TCRa constant region gene comprising SEQ ID NO: 5 comprises a first subunit and a second subunit, wherein the first subunit comprises: (a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more in sequence identity with residues 198-344 of SEQ ID NO: 7 or 8; and (b) a HVR1 region having at least 80%, at least 85%, at least 90%, at least 95%, or more in sequence identity with the amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7 or 8, and comprising residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 7 or 8; and wherein the second subunit comprises: (a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more in sequence identity with residues 7-153 of SEQ ID NO: 7 or 8; and (b) a HVR2 region having at least 80%, at least 85%, at least 90%, at least 95%, or more in sequence identity with the amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7 or 8, and comprising residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 7 or 8. In these embodiments, the HVR2 region can further comprise residues corresponding to residues 48, 50, 71, 72, and 73 of SEQ ID NO: 7 and / or residues corresponding to residues 48 and 50 of SEQ ID NO: 8.

[0051] In particular embodiments, the engineered meganuclease recognizing and cleaving a recognition sequence comprising SEQ ID NO: 5 within a human TCRa constant region gene comprises a first subunit and a second subunit, wherein the first subunit comprises: (a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more in sequence identity to residues 198-344 of SEQ ID NO: 7 or 8; and (b) a HVR1 region having at least 80%, at least 85%, at least 90%, at least 95%, or more in sequence identity to the amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7 or 8, and comprising residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 7 or 8; wherein the second subunit comprises: (a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more in sequence identity to residues 7-153 of SEQ ID NO: 7 or 8; and (b) a HVR2 region having at least 80%, at least 85%, at least 90%, at least 95%, or more in sequence identity to the amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7 or 8, and comprising residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 48, 50, 68, 70, 71, 72, 73, 75, and 77 of SEQ ID NO: 7 or 8.

[0052] In other embodiments, the engineered meganuclease recognizing and cleaving a recognition sequence comprising SEQ ID NO: 5 within a human TCRa constant region gene comprises a first subunit and a second subunit, wherein the first subunit comprises: (a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more in sequence identity to residues 198-344 of SEQ ID NO: 7 or 8; (b) a HVR1 region having the amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7 or 8; wherein the second subunit comprises: (a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more in sequence identity to residues 7-153 of SEQ ID NO: 7 or 8; and (b) a HVR2 region having the amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7 or 8.

[0053] In another aspect, the present application provides a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein.

[0054] In some embodiments, the polynucleotide is an mRNA.

[0055] In other embodiments, the mRNA is a polycistronic mRNA encoding an engineered meganuclease described herein and at least one additional polypeptide or nucleic acid.

[0056] In another aspect, the application provides a recombinant DNA construct comprising a polynucleotide described herein.

[0057] In certain embodiments, the recombinant DNA construct encodes a viral vector. In particular embodiments, the viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an adeno-associated viral (AAV) vector. In specific embodiments, the viral vector is a recombinant AAV vector.

[0058] In another aspect, the application provides a viral vector comprising a polynucleotide described herein.

[0059] In certain embodiments, the viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an AAV vector. In particular embodiments, the viral vector is a recombinant AAV vector.

[0060] In another aspect, the application provides a method of producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted into a chromosome of the eukaryotic cell. The method comprises introducing into the eukaryotic cell one or more nucleic acids comprising: (a) a first nucleic acid encoding an engineered meganuclease described herein, wherein the engineered meganuclease is expressed in the eukaryotic cell; and (b) a second nucleic acid comprising the sequence of interest; wherein the engineered meganuclease generates a cleavage site in the chromosome at a recognition sequence comprising SEQ ID NO: 5; and wherein the sequence of interest is inserted into the chromosome at the cleavage site.

[0061] In certain embodiments of the method, the second nucleic acid further comprises sequences homologous to sequences flanking the cleavage site, and the sequence of interest is inserted at the cleavage site by homologous recombination.

[0062] In certain embodiments of the method, the second nucleic acid does not comprise sequences homologous to sequences flanking the cleavage site, and the sequence of interest is inserted at the cleavage site by non-homologous insertion.

[0063] In certain embodiments of the method, the cell surface expression of an endogenous T cell receptor (e.g., alpha / beta T cell receptor) is reduced compared to an unmodified control cell.

[0064] In some embodiments of the method, the eukaryotic cell is a human T cell or a cell derived therefrom, or a human NK cell or a cell derived therefrom.

[0065] In some embodiments of the method, the sequence of interest comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor. In particular embodiments of the method, the chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand-binding domain specific for a tumor-specific antigen.

[0066] In some embodiments of the method, at least the first nucleic acid is introduced into the eukaryotic cell via mRNA.

[0067] In certain embodiments of the method, at least the second nucleic acid is introduced into the eukaryotic cell via a viral vector. In particular embodiments of the method, the viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an AAV vector. In specific embodiments of the method, the viral vector is a recombinant AAV vector.

[0068] In another aspect, the application provides a method of producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted into a chromosome of the eukaryotic cell. The method comprises: (a) introducing into the eukaryotic cell an engineered meganuclease described herein; and (b) introducing into the eukaryotic cell a nucleic acid comprising the sequence of interest; wherein the engineered meganuclease generates a cleavage site at a recognition sequence of SEQ ID NO: 5 in the chromosome; and wherein the sequence of interest is inserted into the chromosome at the cleavage site.

[0069] In certain embodiments of the method, the nucleic acid further comprises a sequence homologous to a sequence flanking the cleavage site, and the sequence of interest is inserted at the cleavage site by homologous recombination.

[0070] In certain embodiments of the method, the nucleic acid does not comprise a sequence homologous to a sequence flanking the cleavage site, and the sequence of interest is inserted at the cleavage site by non-homologous insertion.

[0071] In certain embodiments of the method, the cell surface expression of an endogenous T cell receptor (e.g., an alpha / beta T cell receptor) is reduced compared to an unmodified control cell.

[0072] In some embodiments of the method, the eukaryotic cell is a human T cell or a cell derived therefrom, or a human NK cell or a cell derived therefrom.

[0073] In some embodiments of the method, the sequence of interest comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor. In particular embodiments of the method, the chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand-binding domain specific for a tumor-specific antigen.

[0074] In certain embodiments of the method, the nucleic acid is introduced into the eukaryotic cell by a viral vector. In particular embodiments of the method, the viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an AAV vector. In specific embodiments of the method, the viral vector is a recombinant AAV vector.

[0075] In another aspect, the application provides a method of producing a genetically modified eukaryotic cell by disrupting a target sequence in a chromosome of the eukaryotic cell. The method comprises introducing into the eukaryotic cell a nucleic acid encoding an engineered meganuclease as described herein, wherein the engineered meganuclease is expressed in the eukaryotic cell, and wherein the engineered meganuclease produces a cleavage site in the chromosome at a recognition sequence comprising SEQ ID NO: 5, and wherein the target sequence is disrupted by non-homologous end joining at the cleavage site.

[0076] In certain embodiments of the method, the cell surface expression of an endogenous T cell receptor (e.g., an alpha / beta T cell receptor) is reduced compared to an unmodified control cell.

[0077] In some embodiments of the method, the eukaryotic cell is a human T cell or a cell derived therefrom, or a human NK cell or a cell derived therefrom.

[0078] In some embodiments of the method, the nucleic acid is introduced into the eukaryotic cell by mRNA.

[0079] In another aspect, the application provides a method of producing a genetically modified eukaryotic cell by disrupting a target sequence in a chromosome of the eukaryotic cell. The method comprises introducing into the eukaryotic cell an engineered meganuclease as described herein, wherein the engineered meganuclease produces a cleavage site in the chromosome at a recognition sequence comprising SEQ ID NO: 5, and wherein the target sequence is disrupted by non-homologous end joining at the cleavage site.

[0080] In certain embodiments of the method, the cell surface expression of an endogenous T cell receptor (e.g., an alpha / beta T cell receptor) is reduced compared to an unmodified control cell.

[0081] In some embodiments of the method, the eukaryotic cell is a human T cell or a cell derived therefrom, or a human NK cell or a cell derived therefrom.

[0082] In another aspect, the present application provides a genetically modified eukaryotic cell comprising in its genome a modified human T cell receptor alpha constant region gene, wherein the modified human T cell receptor alpha constant region gene comprises an exogenous sequence of interest inserted in exon 1 within SEQ ID NO: 5 within the T cell receptor alpha constant region, and wherein the genetically modified eukaryotic cell is made by the methods described herein using the engineered meganuclease described herein.

[0083] In certain embodiments, the genetically modified eukaryotic cell is a genetically modified human T cell or a cell derived therefrom, or a genetically modified human NK cell or a cell derived therefrom.

[0084] In certain embodiments, the sequence of interest comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor. In particular embodiments, the chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand binding domain specific for a tumor specific antigen.

[0085] In particular embodiments, the cell surface expression of an endogenous T cell receptor (e.g., alpha / beta T cell receptor) is reduced on the genetically modified eukaryotic cell compared to an unmodified control cell.

[0086] In particular embodiments, the genetically modified eukaryotic cell comprises a reduced off-target effect of the engineered meganuclease compared to TRC 1-2x.87EE meganuclease as set forth in SEQ ID NO: 9, and / or a reduced duration of the engineered meganuclease in the cell.

[0087] In another aspect, the present application provides a genetically modified eukaryotic cell comprising a chromosome having a disrupted target sequence at a recognition sequence comprising SEQ ID NO: 5, wherein the target sequence is disrupted by non-homologous end joining at the cleavage site, and wherein the genetically modified eukaryotic cell is made by the methods described herein using the engineered meganuclease described herein.

[0088] In certain embodiments, the genetically modified eukaryotic cell is a genetically modified human T cell or a cell derived therefrom, or a genetically modified human NK cell or a cell derived therefrom.

[0089] In particular embodiments, the cell surface expression of an endogenous T cell receptor (e.g., alpha / beta T cell receptor) is reduced on the genetically modified eukaryotic cell when compared to an unmodified control cell.

[0090] In particular embodiments, the genetically modified eukaryotic cell comprises reduced off-target effects of the engineered meganuclease, and / or reduced persistence in the cell compared to a TRC 1-2x.87EE meganuclease as set forth in SEQ ID NO: 9.

[0091] In another aspect, the application provides a population of genetically modified eukaryotic cells comprising a plurality of genetically modified eukaryotic cells described herein.

[0092] In some embodiments, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of the cells in the population are genetically modified eukaryotic cells described herein.

[0093] In particular embodiments, the genetically modified eukaryotic cells of the population are genetically modified human T cells or cells derived therefrom, or genetically modified NK cells or cells derived therefrom.

[0094] In certain embodiments, the genetically modified eukaryotic cells of the population comprise a cell surface chimeric antigen receptor or an exogenous T cell receptor. In some of these embodiments, the chimeric antigen receptor or exogenous T cell receptor comprises an extracellular ligand binding domain specific for a tumor specific antigen.

[0095] In particular embodiments, the genetically modified eukaryotic cells of the population have reduced cell surface expression of endogenous T cell receptors (e.g., alpha / beta T cell receptors) compared to unmodified control cells.

[0096] In another aspect, the application provides a pharmaceutical composition useful for treating a disease in a subject in need thereof, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a therapeutically effective amount of a genetically modified eukaryotic cell or population thereof as described herein.

[0097] In certain embodiments, the genetically modified eukaryotic cell is a genetically modified human T cell or cell derived therefrom, or a genetically modified NK cell or cell derived therefrom, or the population consists of genetically modified human T cells or cells derived therefrom or genetically modified NK cells or cells derived therefrom.

[0098] In some embodiments, the exogenous sequence of interest present in the genetically modified T cell or population thereof comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor. In certain particular embodiments, the chimeric antigen receptor or exogenous T cell receptor comprises an extracellular ligand binding domain specific for a tumor specific antigen.

[0099] In some embodiments, the cell surface expression of an endogenous T cell receptor (e.g., alpha / beta T cell receptor) is reduced on the genetically modified eukaryotic cell compared to an unmodified control cell.

[0100] In another aspect, the application provides a lipid nanoparticle or lipid nanoparticle formulation comprising an mRNA encoding at least one engineered meganuclease described herein. In some embodiments, the lipid nanoparticle has a composition that increases delivery and uptake by T cells.

[0101] In another aspect, the application provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a genetically modified eukaryotic cell or population thereof as described herein.

[0102] In some embodiments, the method comprises administering to the subject a pharmaceutical composition described herein.

[0103] In certain embodiments, the method is an immunotherapy for treating a cancer in a subject in need thereof. In some such embodiments, the genetically modified eukaryotic cell is a genetically modified human T cell or a cell derived therefrom, or a genetically modified human NK cell or a cell derived therefrom, and the exogenous sequence of interest present in the genetically modified eukaryotic cell comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor comprising an extracellular ligand binding domain specific for a tumor specific antigen, and the cell surface expression of an endogenous T cell receptor (e.g., alpha- / beta T cell receptor) is reduced on the genetically modified eukaryotic cell compared to an unmodified control cell.

[0104] In some embodiments of the method, the cancer is selected from the group consisting of a carcinoma, a lymphoma, a sarcoma, a blastoma, and a leukemia.

[0105] In certain embodiments of the method, the cancer is selected from the group consisting of a cancer of B-cell origin, a breast cancer, a gastric cancer, a neuroblastoma, an osteosarcoma, a lung cancer, a melanoma, a prostate cancer, a colon cancer, a renal cell carcinoma, an ovarian cancer, a rhabdomyosarcoma, a leukemia, and a Hodgkin’s lymphoma.

[0106] In particular embodiments of the method, the cancer of B-cell origin is selected from the group consisting of a B-lineage acute lymphoblastic leukemia, a B-cell chronic lymphocytic leukemia, a B-cell non-Hodgkin’s lymphoma, and a multiple myeloma.

[0107] In particular embodiments of the method, the subject can be a mammal, e.g., a human.

[0108] In another aspect, the present application provides a genetically modified cell or population thereof as described herein for use as a medicament. The present application also provides the use of a genetically modified cell or population thereof as described herein in the manufacture of a medicament for treating a disease in a subject in need thereof. In one such aspect, the medicament can be for treating cancer.

[0109] In another aspect, the present application provides a genetically modified cell or population thereof as described herein for use in the treatment of a disease, preferably for use in the treatment of cancer. BRIEF DESCRIPTION OF DRAWINGS

[0110] Figure 1 . TRC 1-2 recognition sequence in the human T cell receptor alpha constant gene. The TRC 1-2 recognition sequence targeted by the engineered meganucleases of the present application comprises two recognition half-sites. Each recognition half-site comprises 9 base pairs separated by a 4 base pair central sequence. The TRC 1-2 recognition sequence (SEQ ID NO: 5) comprises two recognition half-sites referred to as TRC1 and TRC2.

[0111] Figure 2 . The engineered meganucleases of the present application comprise two subunits, wherein the first subunit comprising the HVR1 region binds to the first recognition half-site (e.g., TRC1) and the second subunit comprising the HVR2 region binds to the second recognition site half-site (e.g., TRC2). In embodiments where the engineered meganuclease is a single-chain meganuclease, the first subunit comprising the HVR1 region can be positioned as the N-terminal or C-terminal subunit. Likewise, the second subunit comprising the HVR2 region can be positioned as the N-terminal or C-terminal subunit.

[0112] Figure 3A schematic of a reporter assay in CHO cells for evaluating engineered meganucleases of the application. A CHO cell line was generated in which a reporter cassette was stably integrated into the genome of the cell. The reporter cassette contains, in 5' to 3' order: an SV40 early promoter; 5' flanking 2 / 3 of a GFP gene; a recognition sequence for an engineered meganuclease of the application (e.g., a TRC 1-2 recognition sequence); a recognition sequence for a CHO 23 / 24 meganuclease (WO / 2012 / 167192); and 3' flanking 2 / 3 of a GFP gene. Cells stably transfected with this cassette do not express GFP in the absence of a DNA break inducer. The meganucleases are introduced by transduction of plasmid DNA or mRNA encoding each meganuclease. When a DNA break is induced at either meganuclease recognition sequence, the duplicated regions of the GFP gene recombine with each other to produce a functional GFP gene. The percentage of cells expressing GFP can then be determined by flow cytometry as an indirect measure of the genomic cleavage frequency of the meganuclease.

[0113] Figure 4 Efficiency of engineered meganucleases to recognize and cleave TRC 1-2 recognition sequences in CHO cell reporter assays. TRC 1-2 L.1592, TRC 1-2 L.1775, and TRC 1-2 L.1843 meganucleases were engineered to target the TRC 1-2 recognition sequence (SEQ ID NO: 5) and were screened for efficacy in CHO cell reporter assays. Results shown provide the percentage of cells observed expressing GFP, which indicates the efficacy of each meganuclease to cleave either the target recognition sequence or the CHO 23 / 24 recognition sequence. A negative control (bs) and a first generation TRC 1-2 x.87EE were further included in the assays for comparison. A) CHO reporter assay to evaluate TRC 1-2 L.1592. B) CHO reporter assay to evaluate TRC 1-2 L.1775. C) CHO reporter assay to evaluate TRC 1-2 L.1843.

[0114] Figure 5Efficiency of engineered meganucleases to recognize and cleave TRC Offl recognition sequence (SEQ ID NO: 16) and TRC Off2 recognition sequence (SEQ ID NO: 17) in CHO cell reporter assays. mRNA encoding the TRC 1-2 meganucleases of the application were transfected into CHO reporter cells containing an anti-selectable Offl recognition sequence or Off2 recognition sequence between direct repeats of GFP, as well as a CHO 23-24 recognition sequence. In each assay, a second generation meganuclease was compared to a first generation TRC 1-2 x.87EE meganuclease. A) Cleavage of off-target recognition sequences by TRC 1-2 L.1592 and TRC 1-2 x.87EE. B) Cleavage of off-target recognition sequences by TRC 1-2 L.1775 and TRC 1-2 x.87EE. C) Cleavage of off-target recognition sequences by TRC 1-2 L.1843 and TRC 1-2 x.87EE.

[0115] Figure 6 Efficiency of engineered meganucleases to recognize and cleave TRC 1-2 recognition sequence in CHO cell reporter assays. TRC 1-2 x.87EE (first generation), TRC 1-2 L.1108 (intermediate) and TRC 1-2 L.1469 (intermediate) meganucleases were engineered to target the TRC 1-2 recognition sequence (SEQ ID NO: 5) and screened for efficacy in CHO cell reporter assays at days 2, 5 and 7 post-nucleofection to determine toxicity. Results shown provide the percentage of GFP-expressing cells observed over the 7-day analysis period, indicating the efficacy of each meganuclease to cleave the target recognition sequence or the CHO 23 / 24 recognition sequence as a function of time.

[0116] Figure 7 Efficiency of engineered meganucleases to recognize and cleave TRC 1-2 recognition sequence in CHO cell reporter assays. Second generation TRC 1-2 L.1592, TRC 1-2 L.1775 and TRC 1-2 L.1843 meganucleases were optimized to target the TRC 1-2 recognition sequence (SEQ ID NO: 5) and screened for efficacy in CHO cell reporter assays at days 2, 5 and 7 post-nucleofection to determine toxicity. The analysis also included the first generation TRC 1-2 x.87EE meganuclease and the intermediate TRC 1-2 L.1469 meganuclease for comparison. Results shown provide the percentage of GFP-expressing cells observed over the 7-day analysis period, indicating the efficacy of each meganuclease to cleave the target recognition sequence or the CHO 23 / 24 recognition sequence as a function of time.

[0117] Figure 8 Efficiency of engineered meganucleases to recognize and cleave TRC Offl and Off2 recognition sequences in CHO cell reporter assays. First generation TRC 1-2x.87EE meganuclease, intermediate TRC 1-2L.1469 meganuclease and second generation TRC 1-2L.1592, TRC 1-2L.1775 and TRC 1-2L.1843 meganucleases were screened for efficacy in CHO GFP reporter cells containing the TRC Offl (SEQ ID NO: 16) or Off2 (SEQ ID NO: 17) recognition sequences at days 2, 5 and 7 post-nucleofection to determine toxicity. Results shown provide the percentage of GFP expressing cells observed over a 7 day analysis period. A) Cleavage of Offl recognition sequence. B) Cleavage of Off2 recognition sequence.

[0118] Figure 9 Graphical visualization of oligonucleotide capture data as a measure of the number of potential effective off-target sites. Each off-target cleavage by a particular nuclease is plotted based on the number of unique sequence reads of the probe oligonucleotide captured at that site. For each meganuclease tested (circled), the site (i.e. TRC 1-2 recognition sequence) is expected to have the highest read count.

[0119] Figure 10 Graphical visualization of oligonucleotide capture data with off-target sites plotted by their aligned read counts on the X-axis and the number of mismatched base pairs compared to the expected site represented by color, with darker colors indicating closer overall match between the off-target and expected binding site. Boxes indicate regions of highest confidence.

[0120] Figure 11 Table summarizing in vitro analysis of CAR T cells generated using the first generation TRC 1-2x.87EE meganuclease, intermediate TRC 1-2L.1469 meganuclease or second generation TRC 1-2L.1592, TRC 1-2L.1775 and TRC 1-2L.1843 meganucleases. Meganucleases were screened for gene editing efficiency, post-editing expansion and differentiation potential. CAR T cells were prepared from cells obtained from three different healthy human donors and experiments were performed by three different operators.

[0121] Figure 12 Graphical visualization of oligonucleotide capture data generated from T cell populations obtained from three different healthy human donors.

[0122] Figure 13In vitro analysis of CAR T cells generated using first generation TRC 1-2x.87EE meganuclease or second generation TRC 1-2L.1592, TRC 1-2L.1775 and TRC 1-2L.1843 meganucleases. A) Total number of cells at day 0, 4 and 8 post-editing. B) Total number of edited cells (i.e. TCR negative) at day 0, 4 and 8 post-editing. C) Total number of TCR negative / CAR positive cells at day 0, 4 and 8 post-editing.

[0123] Figure 14 CAR T cell expansion after co-culture with antigen-bearing target cells. CAR T cells were evaluated for expansion after 5 days of co-culture with CD19+ tumor lines Raji or Nalm6 at E:T ratios of 1 : 1 and 1 :2. Cell input numbers are identified by dashed lines.

[0124] Figure 15 CAR T cell expansion after co-culture with antigen-bearing target cells. CAR T cells were evaluated for expansion after 5 days of co-culture with Raji CD19+ tumor cell line at E:T of 1 :2. A) Total number of CAR positive cells in culture after co-culture with Raji cells. B) Total number of CD19 positive cells remaining in culture after co-culture of CAR T cells with Raji cells.

[0125] Figure 16 CAR T cell cytokine secretion into culture supernatant after 2 days of co-culture with antigen-bearing target cells. CAR T cells were evaluated for cytokine secretion after co-culture with CD19+ tumor cell lines Raji or Nalm6 at E:T ratios of 1 : 1 and 1 :2. K562 myeloid leukemia cells, which are CD19 negative, were used as a control. A) IL-2 secretion. B) TNF-a secretion. C) INF-g secretion. D) Granzyme B secretion. E) Perforin secretion.

[0126] Figure 17 Western blot analysis of meganuclease expression in CAR T cells. Cells were electroporated with mRNA encoding TRC 1-2x.87EE or TRC 1-2L.1592 meganucleases and then transduced with a recombinant AAV6 vector carrying a donor template encoding an anti-CD19 CAR designed for insertion at the TRC 1-2 site. Meganuclease protein expression was determined by western blot analysis at 6 hours, 24 hours, 48 hours, 96 hours and 168 hours post-electroporation. Mock cells from the same donor were activated and cultured in the same media as the nuclease-treated groups and harvested at 24 hours post-electroporation of the nuclease-treated groups.

[0127] Figure 18. Total number of viable cells at day 0, 3, 8 (before and after CD3 positive cell depletion), and 13 of a large scale CAR T manufacturing process run using TRC 1-2x.87EE or TRC 1-2L.1592.

[0128] Figure 19 . Total number of viable CD3 negative cells at day 8 of a large scale CAR T manufacturing process run using TRC 1-2x.87EE or TRC 1-2L.1592.

[0129] Figure 20 . Percentage of CD3 negative cells that are CAR positive at day 8 (before and after CD3 positive cell depletion) and day 13 of a large scale CAR T manufacturing process run using TRC 1-2x.87EE or TRC 1-2L.1592.

[0130] Sequence Description

[0131] SEQ ID NO: 1 sets forth the amino acid sequence of a wild-type I-Crel meganuclease from Chlamydomonas reinhardtii.

[0132] SEQ ID NO: 2 sets forth the amino acid sequence of a LAGLIDADG motif.

[0133] SEQ ID NO: 3 sets forth the nucleic acid sequence of a human T cell receptor alpha constant region gene (NCBI Gene ID No. 28755).

[0134] SEQ ID NO: 4 sets forth the amino acid sequence of a polypeptide encoded by a human T cell receptor alpha constant region gene.

[0135] SEQ ID NO: 5 sets forth the nucleic acid sequence of the sense strand of a TRC 1-2 recognition sequence.

[0136] SEQ ID NO: 6 sets forth the nucleic acid sequence of the anti-sense strand of a TRC 1-2 recognition sequence.

[0137] SEQ ID NO: 7 sets forth the amino acid sequence of a TRC 1-2L.1592 meganuclease.

[0138] SEQ ID NO: 8 sets forth the amino acid sequence of a TRC 1-2L.1775 meganuclease.

[0139] SEQ ID NO: 9 sets forth the amino acid sequence of a TRC 1-2x.87EE meganuclease.

[0140] SEQ ID NO: 10 sets forth the amino acid sequence of the TRC 1-2L.1592 meganuclease TRC1 binding subunit.

[0141] SEQ ID NO: 11 sets forth the amino acid sequence of the TRC 1-2L.1775 meganuclease TRC1 binding subunit.

[0142] SEQ ID NO: 12 sets forth the amino acid sequence of the TRC 1-2x.87EE meganuclease TRC1 binding subunit.

[0143] SEQ ID NO: 13 sets forth the amino acid sequence of the TRC 1-2L.1592 meganuclease TRC2 binding subunit.

[0144] SEQ ID NO: 14 sets forth the amino acid sequence of the TRC 1-2L.1775 meganuclease TRC2 binding subunit.

[0145] SEQ ID NO: 15 sets forth the amino acid sequence of the TRC 1-2x.87EE meganuclease TRC2 binding subunit.

[0146] SEQ ID NO: 16 sets forth the nucleic acid sequence of the Offl recognition sequence.

[0147] SEQ ID NO: 17 sets forth the nucleic acid sequence of the Off2 recognition sequence.

[0148] SEQ ID NO: 18 sets forth the amino acid sequence of the polypeptide linker. DETAILED DESCRIPTION

[0149] 1.1 References and Definitions

[0150] The patents and scientific literature referred to herein establish the knowledge that is available to those with skill in the art. U.S. and non-U.S. published patents, published U.S. and non-U.S. and PCT applications, co-owned and co-pending unpublished U.S. patent applications, published foreign applications, and scientific, technical and medical publications, including GenBank database sequences, public domain gene and protein database accession numbers or codes (and the nucleic and / or amino acid sequences associated therewith), cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.

[0151] The application can be embodied in various forms and should not be construed as limited to the embodiments set forth in this document. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, variations and additions to the described embodiments can be apparent to those of ordinary skill in the art in view of the foregoing disclosure, which do not depart from the scope of the application.

[0152] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs as of the priority date. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0153] All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0154] As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a plurality of cells.

[0155] As used herein, and unless explicitly stated otherwise, the word “or” is used in its inclusive sense (i.e., “and / or”), not its exclusive sense (i.e., either / or).

[0156] As used herein, the term “endonuclease” refers to an enzyme that cleaves a phosphodiester bond within a polynucleotide chain.

[0157] As used herein, the term “cleavage” or “cleave” with respect to double-stranded DNA refers to the hydrolysis of a phosphodiester bond within the recognition sequence backbone within a target sequence mediated by an endonuclease, which results in a double-stranded break within the target sequence, referred to herein as a “cleavage site.” Depending on the endonuclease, cleavage can result in double-stranded fragments with blunt ends or fragments with 5’ or 3’ base overhangs.

[0158] As used herein, the term "meganuclease" refers to an endonuclease that binds double stranded DNA at a recognition sequence that is greater than 12 base pairs. In some embodiments, the recognition sequence of a meganuclease of the present disclosure is 22 base pairs. The meganuclease can be an endonuclease derived from I-Crel, and can refer to an engineered variant of I-Crel that has been modified with respect to, for example, DNA binding specificity, DNA cleavage activity, DNA binding affinity, or dimerization properties relative to native I-Crel. Methods of producing such modified variants of I-Crel are known in the art (e.g., WO 2007 / 047859, the entire contents of which are incorporated herein by reference). As used herein, the meganuclease binds double stranded DNA as a heterodimer. The meganuclease can also be a "single-chain meganuclease," in which a pair of DNA binding domains are joined into a single polypeptide using a peptide linker. The term "homing endonuclease" is synonymous with the term "meganuclease." The meganucleases of the present disclosure are substantially non-toxic when expressed in the target cells described herein, particularly in human T cells, such that the cells can be transfected and maintained at 37°C without observing a significant deleterious effect on overall cell viability or a significant reduction in meganuclease cleavage activity when measured using the methods described herein.

[0159] As used herein, the term "single-chain meganuclease" refers to a polypeptide comprising a pair of nuclease subunits connected by a linker, such that the subunits functionally interact like a heterodimer to cleave a double stranded recognition site. The single-chain meganuclease has the following organization: N-terminal subunit - linker - C-terminal subunit. The two meganuclease subunits are typically different in amino acid sequence, and recognize non-identical DNA half-sites within the recognition sequence. Thus, the single-chain meganuclease typically cleaves a pseudo-palindromic or non-palindromic recognition sequence. The single-chain meganuclease can be referred to as a "single-chain heterodimer" or "single-chain heterodimer meganuclease," although it is not actually dimeric. For clarity, the term "meganuclease" can refer to a dimeric or single-chain meganuclease, unless otherwise specified.

[0160] As used herein, the term “linker” refers to an exogenous peptide sequence used to join two meganuclease subunits into a single polypeptide. The linker can have a sequence found in a natural protein, or can be an artificial sequence not found in any natural protein. The linker can be flexible and lack secondary structure, or can tend to form a specific three-dimensional structure under physiological conditions. The linker can include, but is not limited to, any of the linkers encompassed by U.S. Patent Nos. 8,445,251, 9,340,777, 9,434,931, and 10,041,053, the entire contents of which are incorporated herein by reference. In some embodiments, the linker can have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 18, which shows residues 154-195 of SEQ ID NO: 7 or 8. In some embodiments, the linker can have an amino acid sequence comprising SEQ ID NO: 18, which shows residues 154-195 of SEQ ID NO: 7 or 8.

[0161] As used herein, the term “recombinant” or “engineered” with respect to a protein refers to an altered amino acid sequence due to the application of genetic engineering techniques to the nucleic acid encoding the protein and the cell or organism in which the protein is expressed. With respect to a nucleic acid, the term “recombinant” or “engineered” refers to an altered nucleic acid sequence due to the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques; transfection, transformation, and other gene transfer techniques; homologous recombination; site-directed mutagenesis; and gene fusion. A protein having the same amino acid sequence as a naturally occurring protein but produced by cloning and expression in a heterologous host is not considered recombinant according to this definition.

[0162] As used herein, the term “wild type” refers to the most common naturally occurring allele (i.e., polynucleotide sequence) in a population of alleles of the same type of gene, wherein the polypeptide encoded by the wild type allele has its original function. The term “wild type” also refers to the polypeptide encoded by the wild type allele. The wild type allele (i.e., polynucleotide) and polypeptide are distinguishable from a mutant or variant allele and polypeptide, which comprise one or more mutations and / or substitutions relative to the wild type sequence. While a wild type allele or polypeptide can confer a normal phenotype in an organism, a mutant or variant allele or polypeptide can confer an altered phenotype in some cases. A wild type nuclease is distinguishable from a recombinant or non-naturally occurring nuclease. The term “wild type” can also refer to a cell, organism, and / or subject having a wild type allele of a particular gene, or a cell, organism, and / or subject for comparison purposes.

[0163] As used herein, the term "genetically modified" refers to a cell or organism in which or in an ancestor of which a genomic DNA sequence has been intentionally modified by recombinant techniques. As used herein, the term "genetically modified" encompasses the term "transgenic."

[0164] As used herein with respect to recombinant proteins, the term "modification" refers to any insertion, deletion, or substitution of an amino acid residue in a recombinant sequence relative to a reference sequence (e.g., wild-type or native sequence).

[0165] As used herein, the term "recognition sequence" or "recognition site" refers to a DNA sequence that is bound and cleaved by an endonuclease. In the case of meganucleases, the recognition sequence comprises a pair of inverted 9 base pair "half-sites" separated by four base pairs. In the case of single-chain meganucleases, the N-terminal domain of the protein contacts the first half-site, while the C-terminal domain of the protein contacts the second half-site. Cleavage by a meganuclease results in a four base pair 3' "overhang." An "overhang" or "sticky end" is a short single-stranded DNA fragment that can result from endonuclease cleavage of a double-stranded DNA sequence. In the case of meganucleases and single-chain meganucleases derived from I-CreI, the overhang comprises bases 10-13 of the 22 base pair recognition sequence.

[0166] As used herein, the term "target site" or "target sequence" refers to a region of chromosomal DNA of a cell that comprises a recognition sequence for a nuclease.

[0167] As used herein, the term "DNA binding affinity" or "binding affinity" refers to the tendency of a meganuclease to non-covalently associate with a reference DNA molecule (e.g., a recognition sequence or an arbitrary sequence). Binding affinity is measured by the dissociation constant, Kd. As used herein, a nuclease has "altered" binding affinity if its Kd for a reference recognition sequence is changed by a statistically significant percentage or a biologically significant amount (e.g., at least 2x or 2x to 10x) relative to a reference nuclease.

[0168] As used herein, the term "specificity" refers to the ability of a nuclease to recognize and cleave a double-stranded DNA molecule only at a particular base pair sequence, referred to as a recognition sequence, or only at a specific set of recognition sequences. The set of recognition sequences share certain conserved positions or sequence motifs, but can be degenerate at one or more positions. A highly specific nuclease is capable of cleaving only one or a very small number of recognition sequences. Specificity can be determined by any method known in the art.

[0169] As used herein, a nuclease has "altered" specificity if it binds to and cleaves a recognition sequence that does not bind and cleave under physiological conditions to a reference nuclease (e.g., wild type), or if the rate of cleavage of the recognition sequence is increased or decreased by a biologically significant amount (e.g., at least 2x or 2x-10x) relative to the reference nuclease.

[0170] In some embodiments, the engineered meganucleases disclosed herein have improved (i.e., increased) specificity for a target recognition sequence comprising SEQ ID NO: 5 (i.e., TRC 1-2) as compared to TRC 1-2x.87EE meganuclease (the amino acid sequence of which is set forth in SEQ ID NO: 9). Thus, in certain embodiments, the engineered meganucleases disclosed herein exhibit reduced off-target cleavage as compared to TRC 1-2x.87EE meganuclease. Off-target cleavage of a meganuclease can be measured using any method known in the art, including, for example, the oligonucleotide capture assay, the T7 endonuclease I (T7E) assay, digital PCR, targeted sequencing of specific off-target sites, exome sequencing, whole genome sequencing, biotinylated in situ break labeling enrichment and next-generation sequencing (BLESS), genome-wide unbiased DSB identification by sequencing (GUIDE-seq), and linear amplification mediated high throughput genome translocation sequencing (LAM-HTGTS) described herein (see, e.g., Zischewski et al. (2017) Biotechnology Advances 35(1): 95-104, the entire contents of which are incorporated by reference in their entirety).

[0171] As used herein, the term "homologous recombination" or "HR" refers to a natural cellular process in which a double-stranded DNA break is repaired using a homologous DNA sequence as a repair template (see, e.g., Cahill et al. (2006), Front. Biosci. 11 : 1958-1976). The homologous DNA sequence can be an endogenous chromosomal sequence delivered to the cell or an exogenous nucleic acid.

[0172] As used herein, the term "non-homologous end joining" or "NHEJ" refers to a natural cellular process in which double-stranded DNA breaks are repaired by direct joining of two non-homologous DNA fragments (see, e.g., Cahill et al. (2006), Front. Biosci. 11 : 1958-1976). DNA repair by non-homologous end joining is error prone and often results in non-templated additions or deletions of DNA sequence at the site of repair. In some cases, cleavage at a target recognition sequence results in NHEJ at the target recognition site. Cleavage of a target site in a coding sequence of a gene by a nuclease and then DNA repair by NHEJ can introduce a mutation (e.g., a frameshift mutation) in the coding sequence that disrupts gene function. Thus, engineered nucleases can be used to effectively knock out a gene in a population of cells. As used herein, "disrupting a target sequence" refers to introducing a mutation (e.g., a frameshift mutation) that interferes with gene function and prevents expression and / or function of the polypeptide / expression product encoded thereby.

[0173] As used herein, a "homology arm" or "sequence homologous to the sequence flanking a meganuclease cleavage site" refers to the sequence flanking the 5' and 3' ends of a nucleic acid molecule that facilitates insertion of the nucleic acid molecule into the cleavage site created by the meganuclease. Typically, the homology arms can be at least 50 base pairs in length, preferably at least 100 base pairs, and up to 2000 base pairs or more, and can have at least 90%, preferably at least 95% or more sequence homology to their corresponding sequence in the genome.

[0174] As used herein, a "chimeric antigen receptor" or "CAR" refers to an engineered receptor that confers or grafts specificity for an antigen onto an immune effector cell (e.g., a human T cell). A chimeric antigen receptor typically comprises at least an extracellular ligand binding domain or portion and an intracellular domain comprising one or more signaling domains and / or costimulatory domains.

[0175] In some embodiments, the extracellular ligand binding domain or portion is in the form of a single chain variable fragment (scFv) derived from a monoclonal antibody, which provides specificity for a particular epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of a cell (e.g., a cancer cell or other pathogenic cell or particle)). In some embodiments, the scFv is linked by a linker sequence. In various embodiments, the extracellular ligand binding domain is specific for any antigen or epitope of interest. In some embodiments, the scFv is murine, humanized, or fully human.

[0176] The extracellular domain of the chimeric antigen receptor can also comprise a self-antigen (see Payne et al. (2016), Science 353(6295): 179-184), which can be recognized by a self-antigen specific B cell receptor on a B lymphocyte, thereby directing the T cell to specifically target and kill self-reactive B lymphocytes in antibody-mediated autoimmune disease. Such CARs can be referred to as chimeric autoantibody receptors (CAARs), and their use is encompassed by the present invention.

[0177] The extracellular domain of the chimeric antigen receptor can also comprise a naturally occurring ligand for an antigen of interest, or a fragment of a naturally occurring ligand that retains the ability to bind an antigen of interest.

[0178] The intracellular stimulatory domain can comprise one or more cytoplasmic signaling domains that will transmit an activation signal to the immune effector cell upon antigen binding. Such cytoplasmic signaling domains can include, but are not limited to, CD3. The intracellular stimulatory domain can also comprise one or more intracellular costimulatory domains that transmit a proliferation and / or cell survival signal upon ligand binding. Such intracellular costimulatory domains can be any known in the art, and can include, but are not limited to, CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, N1, N6, or any combination thereof.

[0179] The chimeric antigen receptor can further comprise other structural elements, including a transmembrane domain linked to the extracellular ligand-binding domain by a hinge or spacer sequence. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane polypeptide can be a subunit of a T cell receptor (i.e., an alpha, beta, gamma, or zeta polypeptide that makes up the CD3 complex), an IL2 receptor p55 (a chain), p75 (beta chain), or gamma chain, an Fc receptor (e.g., Fc gamma receptor III), or a subunit chain of a CD protein (e.g., CD8 alpha chain). Alternatively, the transmembrane domain can be synthetic and can comprise primarily hydrophobic residues (e.g., leucines and valines).

[0180] A hinge region refers to any oligo- or polypeptide having the function of linking a transmembrane domain to an extracellular ligand binding domain. For example, a hinge region can comprise up to 300 amino acids, preferably 10 to 100 amino acids, most preferably 25 to 50 amino acids. A hinge region can be derived from all or a portion of a naturally occurring molecule, for example, from all or a portion of the extracellular region of CD8, CD4, or CD28, or from all or a portion of an antibody constant region. Alternatively, a hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a completely synthetic hinge sequence. In particular examples, a hinge domain can comprise a portion of a human CD8a chain, an FcyRIIIa receptor, or an IgGl.

[0181] As used herein, an "exogenous T cell receptor" or "exogenous TCR" refers to a TCR whose sequence is introduced into the genome of an immune effector cell (e.g., a human T cell) that can or can not endogenously express a TCR. Expression of an exogenous TCR on an immune effector cell can confer specificity for a particular epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of a cancer cell or other disease-causing cell or particle). Such an exogenous T cell receptor can comprise alpha and beta chains, or alternatively, can comprise a gamma chain and a delta chain. Exogenous TCRs useful in the present application can have specificity for any antigen or epitope of interest.

[0182] As used herein, the term "reduced expression" refers to any reduction in the expression of an endogenous T cell receptor (e.g., alpha / beta T cell receptor) on the cell surface of a genetically modified T cell as compared to a control cell. The term reduced can also refer to a reduction in the percentage of cells in a population of cells that express an endogenous polypeptide (i.e., an endogenous T cell receptor) on the cell surface as compared to a control population of cells. Such a reduction can be up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to 100%. Thus, the term "reduced" includes partial knockdown and complete knockdown of an endogenous T cell receptor. Knockout (i.e., complete knockdown) of cell surface expression of an endogenous T cell receptor can result from inactivation of the T cell receptor alpha constant region gene using the engineered meganucleases described herein. The alpha constant region encoded by the T cell receptor alpha constant region gene is necessary for assembly of an endogenous TCR complex on the cell surface. Thus, knockout of the T cell receptor alpha constant region gene using the engineered meganucleases described herein results in knockout of cell surface T cell receptor expression.

[0183] The terms "percent identity," "sequence identity," "percent similarity," "sequence similarity," and the like, as used herein with respect to both amino acid and nucleic acid sequences, refer to the degree of sequence similarity between two sequences, based on sequence alignment which maximizes the number of identical or similar residues between the aligned amino acid residues or nucleotides, and which is a function of the number of identical or similar residues, the total number of residues in the sequences, and the presence or length of any gaps in the sequence alignment. A variety of algorithms and computer programs are available to determine sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), and are described, for example, in Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet. 3:266-272; Madden et al. (1996), Meth. Enzymol. 266:131-141; Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402); Zhang et al. (2000), J. Comput. Biol. 7(1-2):203-14. As used herein, the percent similarity of two amino acid sequences is the score based on the following parameters for the BLASTp algorithm: word length = 3; gap open penalty = -11; gap extension penalty = -1; scoring matrix = BLOSUM62. As used herein, the percent similarity of two nucleic acid sequences is the score based on the following parameters for the BLASTn algorithm: word length = 11; gap open penalty = -5; gap extension penalty = -2; match reward = 1; mismatch penalty = -3.

[0184] As used herein, the term "corresponds to" with respect to modifications of two proteins or amino acid sequences is used to indicate that a particular modification in a first protein is a substitution of the same amino acid residue as in a modification of a second protein, and that the amino acid position in the first protein that is modified corresponds to or aligns with the amino acid position in the second protein that is modified when the two proteins are subjected to a standard sequence alignment (e.g., using the BLASTp program). Thus, if residues X and Y correspond to each other in a sequence alignment, then a modification of residue "X" in the first protein to amino acid "A" will correspond to a modification of residue "Y" in the second protein to amino acid "A", although X and Y can be different numbers.

[0185] As used herein, the term "recognition half-site," "recognition sequence half-site," or simply "half-site" refers to a nucleic acid sequence in a double-stranded DNA molecule that is recognized by a monomer of a homodimeric or heterodimeric meganuclease, or by one subunit of a single-chain meganuclease.

[0186] As used herein, the term "hypervariable region" refers to a local sequence within a meganuclease monomer or subunit that comprises amino acids with relatively high variability. A hypervariable region can comprise about 50-60 contiguous residues, about 53-57 contiguous residues, or preferably about 56 residues. In some embodiments, the residues of a hypervariable region can correspond to positions 24-79 or 215-270 of SEQ ID NO: 7 or 8. A hypervariable region can comprise one or more residues that contact DNA bases in a recognition sequence, and can be modified to alter the base preference of the monomer or subunit. A hypervariable region can also comprise one or more residues that bind to the DNA backbone when the meganuclease binds to a double-stranded DNA recognition sequence. These residues can be modified to alter the binding affinity of the meganuclease to the DNA backbone and the target recognition sequence. In different embodiments of the application, a hypervariable region can comprise 1-20 residues that exhibit variability and can be modified to affect base preference and / or DNA binding affinity. In particular embodiments, a hypervariable region comprises about 15-20 residues that exhibit variability and can be modified to affect base preference and / or DNA binding affinity.

[0187] In some embodiments, the variable residues within a hypervariable region correspond to one or more of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 7 or 8. In some embodiments, the variable residues within a hypervariable region also correspond to one or more of positions 48, 50, 71, 72, and 73 of SEQ ID NO: 7. In some embodiments, the variable residues within a hypervariable region also correspond to one or more of positions 48 and 50 of SEQ ID NO: 8. In some embodiments, the variable residues within a hypervariable region correspond to one or more of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 48, 50, 68, 70, 71, 72, 73, 75, and 77 of SEQ ID NO: 7 or 8.

[0188] In other embodiments, the variable residues within a hypervariable region correspond to one or more of positions 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 7 or 8.

[0189] As used herein, the terms“T cell receptor alpha gene” or“TCR alpha gene” are interchangeable and refer to the locus in a T cell that encodes the T cell receptor alpha subunit. Prior to or after rearrangement, T cell receptor alpha can refer to NCBI Gene ID No. 6955. After rearrangement, the T cell receptor alpha gene comprises an endogenous promoter, rearranged V and J segments, an endogenous splice donor site, an intron, an endogenous splice acceptor site, and a T cell receptor alpha constant region locus comprising exons encoding the subunit.

[0190] As used herein, the terms“T cell receptor alpha constant region” or“TCR alpha constant region” refer to the coding sequence of the T cell receptor alpha gene. The TCR alpha constant region comprises the wild-type sequence identified by NCBI Gen ID NO. 28755 and functional variants thereof.

[0191] The terms“recombinant DNA construct,”“recombinant construct,”“expression cassette,”“expression construct,”“chimeric construct,”“construct,” and“recombinant DNA fragment” are used interchangeably herein and are single- or double-stranded polynucleotides. A recombinant construct comprises an artificial combination of nucleic acid fragments, including but not limited to, regulatory and coding sequences that are not found together in nature. For example, a recombinant DNA construct can comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged to function in a manner that is not naturally occurring. Such constructs can be used, alone or in combination with a vector, to insert the sequence into a host cell.

[0192] As used herein, a“vector” or“recombinant DNA vector” can be a construct comprising a replication system and sequences capable of transcribing and translating polypeptide-encoding sequences in a given host cell. If a vector is used, the choice of vector depends on the method to be used for transforming host cells, as is well known to those skilled in the art. Vectors can include, but are not limited to, plasmid vectors and recombinant viral vectors (e.g., AAV vectors) or any other vector known in the art suitable for delivering a gene encoding a meganuclease of the application to a target cell. The skilled artisan is well aware of the genetic elements that must be present on a vector in order for a host cell comprising any isolated nucleotide or nucleic acid sequence of the application to be successfully transformed, selected, and propagated.

[0193] As used herein, a“vector” can also refer to a viral vector. Viral vectors can include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors (AAV).

[0194] As used herein, "polycistronic" mRNA refers to a single messenger RNA that comprises two or more coding sequences (i.e., cistrons) and encodes more than one protein. A polycistronic mRNA can comprise any element known in the art to allow translation of two or more genes from the same mRNA molecule, including but not limited to IRES elements, T2A elements, P2A elements, E2A elements, and F2A elements.

[0195] As used herein, "human T cell" or "T cell" refers to a T cell isolated from a donor, particularly a human donor. T cells and cells derived therefrom include isolated T cells that have not been passaged in culture, T cells that have been passaged and maintained in cell culture conditions but are not immortalized, and T cells that are immortalized and can be maintained indefinitely in cell culture conditions.

[0196] As used herein, "control" or "control cell" refers to a cell that provides a reference point for measuring changes in the genotype or phenotype of a genetically modified cell. Control cells can include, for example: (a) a wild-type cell, i.e., a cell having the same genotype as the starting material used for a genetic change that genetically modifies a cell; (b) a cell having the same genotype as a genetically modified cell, but that has been transformed with an ineffective construct, i.e., a construct that has no known effect on the trait of interest; or (c) a cell that is genetically identical to a genetically modified cell, but that has not been exposed to a condition or stimulus that would induce an altered genotype or phenotype expression or further genetic modification.

[0197] As used herein, the term "treat" or "treating a subject" refers to administering a genetically modified T cell or population of genetically modified T cells of the present application to a subject having a disease. For example, a subject can have a disease (e.g., cancer), and treatment can denote an immunotherapy for treating the disease. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some aspects, a genetically modified eukaryotic cell or population of genetically modified eukaryotic cells described herein is administered in the form of a pharmaceutical composition of the present application during treatment.

[0198] The term "effective amount" or "therapeutically effective amount" refers to an amount that is sufficient to achieve a beneficial or desired biological and / or clinical result. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity, and the age, weight, physical condition, and responsiveness of the subject to be treated. In particular embodiments, an effective amount of the genetically modified T cell or population of genetically modified T cells of the application or a pharmaceutical composition disclosed herein reduces at least one symptom of a disease in a subject. In those embodiments in which the disease is a cancer, an effective amount of an engineered meganuclease or pharmaceutical composition disclosed herein reduces the proliferation or metastasis level of the cancer, causes partial or complete response or remission of the cancer, or reduces at least one symptom of the cancer in the subject.

[0199] As used herein, the term "cancer" is understood to encompass any neoplastic disease characterized by abnormal and uncontrolled cellular proliferation leading to malignant growths or tumors, whether invasive or metastatic.

[0200] As used herein, the term "carcinoma" refers to a malignant growth composed of epithelial cells.

[0201] As used herein, the term "leukemia" refers to a malignant tumor of the blood-forming organs / systems and is generally characterized by the abnormal proliferation and development of leukocytes and their precursors in the blood and bone marrow.

[0202] As used herein, the term "sarcoma" refers to a tumor composed of substance similar to embryonic connective tissue and is generally composed of compactly packed cells embedded in a fibrillary, heterogeneous, or homogeneous substance.

[0203] As used herein, the term "melanoma" refers to a tumor that arises from the melanocyte system of the skin and other organs.

[0204] As used herein, the term "lymphoma" refers to a group of blood cell tumors that develop from lymphocytes.

[0205] As used herein, the term "blastoma" refers to a type of cancer caused by a malignant tumor in precursor cells or blast cells (immature or embryonic tissue).

[0206] As used herein, recitation of ranges of values of variables is intended to convey that the invention can be practiced with any value within the range. Thus, for inherently discrete variables, the variable can have any integer value within the range, including the endpoints of the range. Similarly, for inherently continuous variables, the variable can have any real value within the range, including the endpoints of the range. For example, and without limitation, if a variable is inherently discrete, a variable described as having a value between 0 and 2 can take on the value of 0, 1, or 2, and if the variable is inherently continuous, the variable can take on the value of 0.0, 0.1, 0.01, 0.001, or any other real number value > 0 and < 2.

[0207] 2.1 Inventive Principle

[0208] The present invention is based, in part, on the discovery of optimized second generation meganucleases having improved properties compared to the parent first generation meganucleases, such as increased (i.e., enhanced) specificity and reduced off-target cleavage, reduced cell persistence following expression from mRNA, improved cell properties when used with human T cells in vitro, improved cell properties when used in large scale CAR T cell manufacturing processes.

[0209] Like the previously described TRC 1-2x.87EE meganuclease, these optimized second generation meganucleases recognize the TRC 1-2 recognition sequence (SEQ ID NO: 5) in exon 1 of the TCRa constant region gene. Cleavage at this recognition sequence can allow for NHEJ at the cleavage site and disrupt expression of the human T cell receptor a chain subunit, resulting in reduced expression and / or function of the T cell receptor at the cell surface. Additionally, cleavage at this recognition sequence can further allow for direct homologous recombination of an exogenous nucleic acid sequence into the TCRa constant region gene. Such exogenous nucleic acid sequences can comprise sequences of interest, such as sequences encoding a chimeric antigen receptor, an exogenous TCR receptor, or any other polypeptide of interest. Thus, the presently disclosed compositions and methods allow for knock-out of endogenous T cell receptors (e.g., a / b T cell receptors) and expression of exogenous nucleic acid sequences (e.g., chimeric antigen receptors or exogenous TCRs). When administered to allogeneic subjects, such cells can exhibit reduced or no induction of graft versus host disease (GVHD).

[0210] 2.2 Recognition and cleavage of TRCs in the T cell receptor α homeostasis region gene 1-2 Optimization of the large-scale kernel for recognizing sequences acid enzyme

[0211] It is known in the art that site-specific nucleases can be used to make DNA breaks in the genome of living cells, and that such DNA breaks can lead to permanent modification of the genome through homologous recombination of the cleaved target site with identical or highly homologous DNA sequences within the genome. Thus, in some embodiments, the present application can be practiced using engineered meganucleases.

[0212] In particular embodiments, the nuclease used to practice the present application is a single-chain meganuclease. Single-chain meganucleases comprise an N-terminal subunit and a C-terminal subunit linked by a linker peptide. Each of the two domains recognizes half of a recognition sequence (i.e., recognizes a half-site), and the site of DNA cleavage is in the middle of the recognition sequence, near the interface of the two subunits. The DNA strand break is offset by four base pairs, such that DNA cleavage by a meganuclease produces a pair of four-base-pair 3' -single-stranded overhangs.

[0213] The meganucleases of the present application have been engineered to recognize and cleave the TRC 1-2 recognition sequence (SEQ ID NO: 5) within exon 1 of the TCRa constant region gene (SEQ ID NO: 3). The engineered meganucleases of the present application comprise a first subunit comprising a first hypervariable (HVR1) region and a second subunit comprising a second hypervariable (HVR2) region. In addition, the first subunit binds a first recognition half-site (i.e., the TRC1 half-site) in the recognition sequence, and the second subunit binds a second recognition half-site (i.e., the TRC2 half-site) in the recognition sequence. In embodiments in which the meganuclease is a single-chain meganuclease, the first subunit and the second subunit can be oriented such that the first subunit comprising the HVR1 region and binding the first half-site is positioned as the N-terminal subunit, and the second subunit comprising the HVR2 region and binding the second half-site is positioned as the C-terminal subunit. In alternative embodiments, the first subunit and the second subunit can be oriented such that the first subunit comprising the HVR1 region and binding the first half-site is positioned as the C-terminal subunit, and the second subunit comprising the HVR2 region and binding the second half-site is positioned as the N-terminal subunit. Table 1 provides exemplary engineered meganucleases that recognize and cleave the TRC 1-2 recognition sequence.

[0214] Table 1. Exemplary engineered meganucleases that recognize and cleave the TRC 1-2 recognition sequence (SEQ ID NO: 5)

[0215]

[0216] *“HVR1 %” and “HVR2 %” represent the amino acid sequence identity between the respective HVR1 region and HVR2 region of each meganuclease and the respective HVR1 region and HVR2 region of the TRC 1-2 x.87EE meganuclease.

[0217] In some embodiments, the presently disclosed engineered meganucleases exhibit at least one optimized property compared to the first generation meganuclease TRC 1-2x.87EE. Such optimized properties include improved (i.e., increased) specificity (resulting in reduced off-target cleavage), reduced persistence in cells after mRNA expression, and enhanced (i.e., increased) cleavage and modification efficiency of the TCRa constant region gene. Thus, in particular embodiments, upon delivery of the presently disclosed engineered meganucleases to a population of eukaryotic cells, they are capable of producing a greater percentage of cells having cleavage and / or modification of the TCRa constant region gene. In some of these embodiments, the population of eukaryotic cells comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more eukaryotic cells comprising a cleavage and / or an insertion / deletion (“indel”) in the TCRa constant region gene. Cleavage and / or modification of the TCRa constant region gene by a meganuclease can be measured using any method known in the art, including T7 endonuclease I assays, digital PCR, mismatch detection assays, mismatch cleavage assays, high resolution melt analysis (HRMA), heteroduplex mobility assays, sequencing, and fluorescent PCR capillary gel electrophoresis (see, e.g., Zischewski et al. (2017) Biotechnology Advances 35(1): 95-104, the entire contents of which are incorporated herein by reference).

[0218] In certain embodiments, the presently disclosed engineered meganucleases exhibit reduced persistence in cells compared to the first generation TRC 1-2x.87EE meganuclease, particularly when introduced as mRNA. Persistence of mRNA or protein within a cell can be measured using any method known in the art, including, but not limited to, RT-PCR, Northern blot analysis, nuclease protection assays, in situ hybridization, immunocytochemistry, immunoblotting, and immunoprecipitation.

[0219] 2.3 Methods for delivering and expressing a wide range of optimized nucleases

[0220] The present disclosure provides methods of generating genetically modified T cells and populations thereof using engineered meganucleases that recognize and cleave a recognition sequence present in the human TCRa constant region gene (SEQ ID NO: 3). T cells can be obtained from a variety of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the disclosure, any number of T cell lines available in the art can be used. In some embodiments of the disclosure, T cells are obtained from a unit of blood collected from a subject using a variety of techniques known to those of skill in the art. In one embodiment, cells from an individual's circulating blood are obtained by apheresis.

[0221] The modified T cell receptor a gene comprises a foreign sequence of interest inserted into the first exon of the TCRa constant region gene (i.e., the target exon) by the double-stranded cleavage of the presently disclosed engineered meganucleases. The cleavage site created by such meganucleases can allow for direct homologous recombination of the foreign sequence of interest into the target exon.

[0222] As used herein, the term "foreign" or "heterologous" in reference to a nucleotide sequence means a sequence that is synthetically made, derived from a foreign substance, or if from the same substance, substantially altered in composition and / or genomic locus from its native form by intentional human intervention.

[0223] In various embodiments, the foreign sequence of interest can comprise a coding sequence for a protein of interest. It is envisioned that the coding sequence can be for any protein of interest.

[0224] In certain embodiments, the foreign sequence of interest comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR). Generally, the CAR of the present disclosure will comprise at least an extracellular domain and an intracellular domain. In some embodiments, the extracellular domain comprises a target-specific binding element, otherwise referred to as a ligand-binding domain or moiety. In some embodiments, the intracellular domain or cytoplasmic domain comprises at least one costimulatory domain and one or more signaling domains, such as CD3 zeta.

[0225] In some embodiments, a CAR useful in the application comprises an extracellular target-specific binding element, otherwise referred to as a ligand binding domain or moiety. The choice of ligand binding domain depends on the type and number of ligands that define the surface of a target cell. For example, a ligand binding domain can be selected to recognize a ligand that serves as a cell surface marker on a target cell that is associated with a particular disease state. Thus, examples of cell surface markers that can serve as ligands for a ligand binding domain in a CAR can include those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells. In some embodiments, a CAR can be engineered to target a tumor-specific antigen of interest by way of engineering a desired ligand binding moiety that specifically binds to an antigen on a tumor cell. In the context of the present disclosure, a "tumor antigen" or "tumor-specific antigen" refers to an antigen common to a particular hyperproliferative disorder, e.g., a cancer.

[0226] In some embodiments, the extracellular ligand binding domain of the CAR is specific for any antigen or epitope of interest, particularly any tumor antigen or epitope of interest. As non-limiting examples, in some embodiments, the antigen of the target is a tumor associated surface antigen such as ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD22, CD30, CD40, CLL-1, the disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, a sphingolipid, a glioma-associated antigen, B human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, survivin and telomerase, prostate carcinoma tumor antigen 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF1)-1, IGF-II, IGF1 receptor, mesothelin, major histocompatibility complex (MHC) molecules presenting tumor specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stroma antigens, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and Al domain of tenascin C (TnC Al) and fibroblast associated protein (fap); lineage- or tissue-specific antigens such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD38, CD123, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), endoglin, major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF 17), CS1; or viral specific surface antigens such as HIV specific antigens (e.g., HIV gpl20), EBV specific antigens, CMV specific antigens, HPV specific antigens (e.g., E6 or E7 oncoproteins), Lasse virus specific antigens, influenza virus specific antigens, and any derivative or variant of these surface markers. In particular embodiments of the disclosure, the ligand binding domain is specific for CD19.

[0227] In some embodiments, the extracellular domain of the chimeric antigen receptor further comprises an autoantigen (see, Payne et al. (2016) Science, Vol. 353(6295): 179-184), which can be recognized by an autoantigen-specific B cell receptor on a B lymphocyte, thereby directing T cell specific targeting and killing of autoreactive B lymphocytes in antibody-mediated autoimmune diseases. Such CARs can be referred to as chimeric autoantibody receptors (CAARs).

[0228] In some embodiments, the extracellular domain of the chimeric antigen receptor can comprise a naturally occurring ligand for the antigen of interest or a fragment of a naturally occurring ligand that retains the ability to bind the antigen of interest.

[0229] In some embodiments, the CAR comprises a transmembrane domain that links the extracellular ligand binding domain or autoantigen to the intracellular signaling and costimulatory domains through a hinge or spacer sequence. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane polypeptide can be a subunit of the T cell receptor (i.e., the a, b, g or z polypeptides that make up the CD3 complex), a subunit chain of the IL2 receptor p55 (a chain), p75 (b chain) or g chain, a subunit chain of the Fc receptor (e.g., Fcy receptor III) or a CD protein (e.g., CD8 a chain). Alternatively, the transmembrane domain can be synthetic and can comprise primarily hydrophobic residues, such as leucine and valine. In particular embodiments, the transmembrane domain is a CD8 a transmembrane polypeptide.

[0230] A hinge region refers to any oligo- or polypeptide that has the function of linking a transmembrane domain to an extracellular ligand binding domain. For example, the hinge region can comprise up to 300 amino acids, preferably 10 to 100 amino acids, most preferably 25 to 50 amino acids. The hinge region can be derived from all or a portion of a naturally occurring molecule, such as all or a portion of the extracellular region of CD8, CD4 or CD28, or all or a portion of an antibody constant region. Alternatively, the hinge region can be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or can be a completely synthetic hinge sequence. In particular embodiments, the hinge domain can comprise a portion of the human CD8 a chain, the FcyRIIIa receptor or IgGl.

[0231] The intracellular signaling domain of a CAR is responsible for activating at least one normal effector function of the cell in which the CAR has been placed and / or activating proliferation and cell survival pathways. The term "effector function" refers to a specialized function of a cell. Effector functions of T cells, for example, can be cytolytic activity or helper activity, including secretion of cytokines. The intracellular signaling domain (e.g., CD3 zeta) can provide an activation signal to the cell in response to binding of the extracellular domain. As discussed, the activation signal can induce effector functions of the cell, such as cytolytic activity or cytokine secretion.

[0232] The intracellular domain of a CAR can include one or more intracellular costimulatory domains that deliver a costimulatory signal following binding of the extracellular domain to promote cell proliferation, cell survival, and / or cytokine secretion. Such intracellular costimulatory domains include those known in the art, such as, but not limited to, N1, N6, CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

[0233] A CAR can be specific for any type of cancer cell. Such cancers can include, but are not limited to, carcinoma, lymphoma, sarcoma, blastoma, leukemia, cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin's lymphoma. In certain embodiments, cancers of B-cell origin include, but are not limited to, B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin's lymphoma, and multiple myeloma.

[0234] The sequence of interest can further encode an exogenous T cell receptor (TCR). Such an exogenous T cell receptor can comprise alpha and beta chains, or alternatively can comprise gamma and delta chains. Exogenous TCRs useful in the present application can be specific for any antigen or epitope of interest.

[0235] In other embodiments, the sequence of interest can encode a wild-type or modified form of an endogenous gene of interest.

[0236] The sequence of interest can comprise elements or peptides known in the art to allow for translation of more than two genes from the same promoter, including but not limited to IRES elements and 2A elements, such as T2A elements, P2A elements, E2A elements, and F2A elements. In particular embodiments, such elements in the exogenous sequence of interest can be located 5' upstream or 3' downstream of the nucleic acid sequence encoding the protein of interest (e.g., a CAR).

[0237] The exogenous sequence of interest described herein can further comprise additional control sequences. For example, the sequence of interest can comprise a homologous recombination enhancer sequence, a Kozak sequence, a polyadenylation sequence, a transcription termination sequence, a selectable marker sequence (e.g., an antibiotic resistance gene), an origin of replication, etc. The sequence of interest described herein can also comprise at least one nuclear localization signal. Examples of nuclear localization signals are known in the art (see, e.g., Lange et al., J. Biol. Chem., 2007, 282:5101-5105).

[0238] The engineered meganucleases of the application can be delivered to a cell in the form of a protein or, preferably, as a nucleic acid encoding the engineered meganuclease. Such a nucleic acid can be DNA (e.g., circular or linearized plasmid DNA or a PCR product) or RNA (e.g., mRNA). For embodiments in which the engineered meganuclease-encoding sequence is delivered in the form of DNA, it should be operably linked to a promoter to facilitate transcription of the meganuclease gene. Mammalian promoters suitable for use in the application include constitutive promoters (e.g., the cytomegalovirus early (CMV) promoter ((Thomsen et al. (1984), Proc Natl Acad Sci USA. 81(3):659-63)) or the SV40 early promoter ((Benoist and Chambon (1981), Nature. 290(5804):304-10))) as well as inducible promoters (e.g., the tetracycline-inducible promoter ((Dingermann et al. (1992), Mol Cell Biol. 12(9):4038-45))). The engineered meganucleases of the application can also be operably linked to synthetic promoters. Synthetic promoters can include, but are not limited to, the JeT promoter (WO 2002 / 012514).

[0239] In some embodiments, mRNA encoding the engineered meganuclease is delivered to the cell, as this reduces the likelihood of integration of the gene encoding the engineered meganuclease into the cell genome. Such mRNA encoding the engineered meganuclease can be produced using methods known in the art, e.g., in vitro transcription. In some embodiments, the mRNA uses 7-methylguanosine, anti-reverse cap analog (ARCA) (US 7,074,596), Analogues such as Cap 1 analogues (Trilink, San Diego, CA) cap the 5' or are enzymatically capped using vaccinia capping enzyme or similar. In some embodiments, the mRNA can be polyadenylated. The mRNA can comprise various 5' and 3' untranslated sequence elements to enhance expression of the encoded engineered meganuclease and / or stability of the mRNA itself. Such elements can include, for example, a post-translational regulatory element (e.g., the woodchuck hepatitis virus post-translational regulatory element). The mRNA can comprise nucleoside analogues or naturally occurring nucleosides, such as pseudouridine, 5-methylcytidine, N6-methyladenosine, 5-methyluridine, or 2-thiouridine. Additional nucleoside analogues include, for example, those described in US 8,278,036.

[0240] In particular embodiments, the mRNA encoding an engineered meganuclease of the application can be a polycistronic mRNA encoding two or more meganucleases that are expressed simultaneously in a cell. The polycistronic mRNA can encode two or more meganucleases that target different recognition sequences in the same target gene. Alternatively, the polycistronic mRNA can encode at least one meganuclease described herein and at least one additional nuclease that targets a separate recognition sequence located in the same gene, or targets a second recognition sequence located in a second gene, thereby creating a cleavage site in both genes. The polycistronic mRNA can comprise any element known in the art to allow translation of two or more genes from the same mRNA molecule (i.e., cistrons), including but not limited to IRES elements, T2A elements, P2A elements, E2A elements, and F2A elements.

[0241] In another particular embodiment, a nucleic acid encoding an engineered meganuclease of the application can be introduced into a cell using a single-stranded DNA template. The single-stranded DNA can further comprise 5' and / or 3' AAV inverted terminal repeat (ITR) sequences upstream and / or downstream of the sequence encoding the engineered meganuclease. In other embodiments, the single-stranded DNA can further comprise 5' and / or 3' homology arms upstream and / or downstream of the sequence encoding the engineered meganuclease.

[0242] In another particular embodiment, a gene encoding a meganuclease of the application can be introduced into a cell using a linearized DNA template. In some examples, the plasmid DNA encoding the meganuclease can be digested with one or more restriction enzymes such that the circular plasmid DNA is linearized prior to being introduced into the cell.

[0243] Purified meganuclease proteins can be delivered into cells to cleave genomic DNA by a variety of different mechanisms known in the art, including those detailed further below, which allow for homologous recombination or non-homologous end joining with a sequence of interest at the cleavage site.

[0244] In some embodiments, the meganuclease protein or DNA / mRNA encoding the meganuclease is conjugated to a cell-penetrating peptide or a targeting ligand to facilitate cellular uptake. Examples of cell-penetrating peptides known in the art include polyarginine (Jearawiriyapaisarn et al. (2008) Mol Ther. 16: 1624-9), TAT peptide from the HIV virus (Hudecz et al. (2005), Med. Res. Rev. 25:679-736), MPG (Simeoni et al. (2003) Nucleic Acids Res. 31 :2717-2724), Pep-1 (Deshayes et al. (2004) Biochemistry 43:7698-7706), and HSV-1 VP-22 (Deshayes et al. (2005) Cell Mol Life Sci. 62: 1839-49). In alternative embodiments, the meganuclease protein or DNA / mRNA encoding the meganuclease is covalently or non-covalently conjugated to an antibody that recognizes a specific cell surface receptor expressed on the target cell, thereby allowing the meganuclease protein / DNA / mRNA to bind to and be internalized by the target cell. Alternatively, the meganuclease protein / DNA / mRNA can be covalently or non-covalently conjugated to a natural ligand (or a portion of a natural ligand) for such a cell surface receptor. (McCall et al. (2014) Tissue Barriers. 2(4):e944449; Dinda et al. (2013) Curr Pharm Biotechnol. 14: 1264-74; Kang et al. (2014) Curr Pharm Biotechnol. 15(3):220-30; Qian et al. (2014) Expert Opin Drug Metab Toxicol. 10(11): 1491-508).

[0245] In some embodiments, the meganuclease protein or DNA / mRNA encoding the meganuclease is covalently or non-covalently coupled to or encapsulated within a nanoparticle using methods known in the art (Sharma, et al. (2014) Biomed Res Int. 2014). Nanoparticles are nanoscale delivery systems with length scales < 1 pm, preferably < 100 nm. Such nanoparticles can be designed using a core composed of metals, lipids, polymers, or biological macromolecules, and multiple copies of the recombinant meganuclease protein, mRNA, or DNA can be attached to or encapsulated within the nanoparticle core. This increases the copy number of protein / mRNA / DNA delivered to each cell, and thus increases the intracellular expression of each engineered meganuclease, maximizing the likelihood of target recognition sequence cleavage. The surface of such nanoparticles can be further modified with polymers or lipids (e.g., chitosan, cationic polymers, or cationic lipids) to form core-shell nanoparticles, the surface of which confers additional functionality to enhance delivery and cellular uptake of the payload (Jian et al. (2012) Biomaterials. 33(30):7621-30). Nanoparticles can additionally be advantageously coupled to targeting molecules to direct the nanoparticle to the appropriate cell type and / or increase the likelihood of cellular uptake. Examples of such targeting molecules include antibodies specific for cell surface receptors and natural ligands (or portions of natural ligands) of cell surface receptors.

[0246] In some embodiments, the meganuclease protein or DNA / mRNA encoding the meganuclease is encapsulated within or complexed using cationic lipids (see, e.g., Lipofectamine TM , Life Technologies Corp., Carlsbad, CA; Zuris et al. (2015) Nat Biotechnol. 33:73-80; Mishra et al. (2011) J Drug Deliv. 2011:863734). Liposome and lipoplex formulations can protect the payload from degradation and facilitate cellular uptake and delivery efficiency by fusing with and / or disrupting the cell membrane of the target cell.

[0247] In some embodiments, the meganuclease protein or DNA / mRNA encoding the meganuclease is encapsulated within a polymeric scaffold (e.g., PLGA) or complexed using a cationic polymer (e.g., PEI, PLL) (Tamboli et al. (2011) Ther Deliv. 2(4):523-536). The polymeric carriers can be designed to provide tunable drug release rates by controlling polymer erosion and drug diffusion, and high drug encapsulation efficiency can provide protection of the therapeutic payload until intracellular delivery to the desired target cell population.

[0248] In some embodiments, the meganuclease protein or DNA / mRNA encoding the recombinant meganuclease is combined with an amphiphilic molecule that self-assembles into micelles (Tong et al. (2007) J Gene Med. 9(11):956-66). Polymeric micelles can include a micellar shell formed by a hydrophilic polymer (e.g., polyethylene glycol) that can prevent aggregation, shield charge interactions, and reduce non-specific interactions.

[0249] In some embodiments, the meganuclease protein or DNA / mRNA encoding the meganuclease is formulated as an emulsion or nanoemulsion (i.e., having an average particle size of less than 1 nm) for administration and / or delivery to target cells. The term "emulsion" refers to, without limitation, any oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil dispersion or droplet (including lipid structures) that can form as a result of the hydrophobic forces that drive polar head groups toward water as a result of driving nonpolar residues (e.g., long hydrocarbon chains) away from water when a water-insoluble phase is mixed with an aqueous phase. These other lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases. Emulsions are composed of an aqueous phase and a lipophilic phase (typically comprising an oil and an organic solvent). Emulsions also often include one or more surfactants. Nanoemulsion formulations are well known, for example, as described in U.S. Patent Application Nos. 2002 / 0045667 and 2004 / 0043041 and U.S. Patent Nos. 6,015,832, 6,506,803, 6,635,676, and 6,559,189, the entire contents of each of which are incorporated herein by reference.

[0250] In some embodiments, the meganuclease protein or DNA / mRNA encoding the meganuclease is covalently linked or non-covalently associated with multifunctional polymer conjugates, DNA dendrimers, and polymeric dendrimers (Mastorakos et al. (2015) Nanoscale. 7(9):3845-56; Cheng et al. (2008) J Pharm Sci. 97(1): 123-43). The generation of dendrimers can control the payload capacity and size, and can provide high drug payload capacity. Moreover, the display of multiple surface groups can be exploited to improve stability, reduce non-specific interactions, and enhance cell-specific targeting and drug release.

[0251] In some embodiments, the genes encoding the meganucleases are delivered using viral vectors. Such vectors are known in the art and include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral (AAV) vectors (reviewed in Vannucci et al. 2013 New Microbiol. 36: 1-22). Recombinant AAV vectors useful in the present application can have any serotype that allows for transduction of the virus into a cell and insertion of the nuclease gene into the cell genome. In particular embodiments, the recombinant AAV vector has the serotype of AAV2 or AAV6, and the AAV vector can also be self-complementary, such that they do not require synthesis of a second strand of DNA in the host cell (McCarty, et al. (2001) Gene Ther. 8: 1248-54).

[0252] If the meganuclease genes are delivered in DNA form (e.g., a plasmid) and / or by a viral vector (e.g., AAV), they must be operably linked to a promoter. In some embodiments, this can be a viral promoter, such as an endogenous promoter from the viral vector (e.g., the LTR of a lentiviral vector) or the well-known cytomegalovirus or SV40 viral early promoters. In preferred embodiments, the meganuclease genes are operably linked to a promoter that preferentially drives gene expression in the target cell (e.g., a T cell).

[0253] The present application is further used to introduce an exogenous sequence of interest into the T cell receptor alpha constant region gene at the TRC 1-2 recognition sequence. In some embodiments, the exogenous sequence of interest comprises 5' and 3' homology arms flanking elements of the insert. Such homology arms have sequence homology to the corresponding sequences upstream 5' and downstream 3' of the nuclease recognition sequence where the cleavage site is generated. Typically, the homology arms can be at least 50 base pairs in length, preferably at least 100 base pairs, and up to 2000 base pairs or more, and can have at least 90%, preferably at least 95% or more sequence homology to their corresponding sequences in the genome.

[0254] The exogenous sequence of interest of the present application can be introduced into a cell by any of the methods discussed above. In particular embodiments, the exogenous sequence of interest is introduced by a viral vector, such as a lentivirus, a retrovirus, an adenovirus, or preferably a recombinant AAV vector. Recombinant AAV vectors useful for introducing an exogenous nucleic acid can be of any serotype that allows for transduction of the virus into a cell and insertion of the exogenous nucleic acid sequence into the genome of the cell. In particular embodiments, the recombinant AAV vector is of the serotype of AAV2 or AAV6. The recombinant AAV vector can also be self-complementary, such that they do not require synthesis of a second strand of DNA in the host cell.

[0255] In another particular embodiment, the exogenous sequence of interest can be introduced into a cell using a single-stranded DNA template. The single-stranded DNA can comprise the exogenous sequence of interest, and in preferred embodiments, can comprise 5' and 3' homology arms to facilitate insertion of the nucleic acid sequence into the meganuclease cleavage site by homologous recombination. The single-stranded DNA can further comprise a 5' AAV inverted terminal repeat (ITR) sequence upstream of 5' homology arm, and a 3' AAV ITR sequence downstream of 3' homology arm.

[0256] In another particular embodiment, the gene encoding the engineered nuclease of the present application and / or the exogenous sequence of interest of the present application can be introduced into a cell by transfection with a linearized DNA template. In some examples, the plasmid DNA can be digested with one or more restriction enzymes, such that the circular plasmid DNA is linearized prior to transfection into the cell.

[0257] The T cells modified by the present application can require activation prior to introduction of the meganuclease and / or exogenous sequence of interest. For example, the T cells can be contacted with anti-CD3 and anti-CD28 antibodies, either soluble or conjugated to a support (i.e., a bead), for a time sufficient to activate the cells.

[0258] The genetically modified cells of the present application can be further modified to express one or more inducible suicide genes, induction of which causes cell death and allows for selective destruction of the cells in vitro or in vivo. In some embodiments, the suicide gene can encode a cytotoxic polypeptide (i.e., a polypeptide with the ability to convert a non-toxic prodrug into a cytotoxic drug) and / or a polypeptide that activates an intracellular cytotoxic gene pathway. That is, a suicide gene is a nucleic acid that encodes a product that causes cell death either by itself or in the presence of other compounds. Representative examples of such suicide genes are the gene encoding the thymidine kinase of the herpes simplex virus. Additional examples are the gene encoding the thymidine kinase of the varicella zoster virus and the bacterial cytosine deaminase gene, which can convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil. Suicide genes also include, by way of non-limiting example, genes encoding caspase-9, caspase-8, or cytosine deaminase. In some examples, caspase-9 can be activated using a specific dimerization chemical inducer (CID). Suicide genes can also encode a polypeptide expressed at the cell surface that sensitizes the cell to a therapeutic and / or cytotoxic monoclonal antibody. In further examples, the suicide gene can encode a recombinant antigenic polypeptide comprising an antigenic motif recognized by the anti-CD20 mAb rituximab and an epitope that allows for selection of cells expressing the suicide gene. See, e.g., the RQR8 polypeptide described in WO2013153391, which comprises two rituximab-binding epitopes and a QBEndlO-binding epitope. For this gene, rituximab can be administered to the subject as needed to induce cell depletion. In further examples, the suicide gene can include a QBEndlO-binding epitope expressed in combination with a truncated EGFR polypeptide.

[0259] Eukaryotic cells modified by the methods and compositions described herein can have reduced expression of endogenous T cell receptors (i.e., a / b T cell receptors) and can optionally further express a protein of interest (e.g., a CAR). Accordingly, the present application further provides a population of eukaryotic cells expressing a protein of interest and not expressing an endogenous T cell receptor (e.g., a / b T cell receptor). For example, the population can include a plurality of genetically modified eukaryotic cells of the present application that express a CAR (i.e., CAR+) or an exogenous T cell receptor (i.e., exoTCR+) and have reduced expression of an endogenous T cell receptor (i.e., TCR-). In various embodiments of the present application, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of the cells in the population are genetically modified eukaryotic cells as described herein. In particular examples, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of the cells included in the population can be TCR- and CAR+.

[0260] In some embodiments, the presently disclosed engineered meganucleases produce a higher percentage of a population of TCR- and CAR+ cells when introduced into a population of cells than when the first generation TCR 1-2x.87EE meganuclease is introduced into a population of cells.

[0261] In addition, cells genetically modified with the presently disclosed engineered meganucleases exhibit improved properties compared to cells genetically modified with the TRC1-2x.87EE meganuclease, including reduced off-target cleavage and effects thereof, reduced persistence of the meganuclease in the cell, enhanced (i.e., increased) CAR T expansion, and lower differentiation. In addition, cell populations that have introduced the presently disclosed meganucleases (or nucleic acids encoding the same) have a greater percentage of modified cells and a greater percentage of less differentiated cells compared to those cell populations in which the TRC1-2x.87EE meganuclease (or nucleic acids encoding the same) was introduced. In particular embodiments, cell populations that have introduced the presently disclosed engineered meganucleases exhibit a higher percentage of central memory T cells (e.g., those expressing CD45RO, CCR7, and CD62L) than cell populations in which the first generation TRC1-2x.87EE meganuclease was introduced.

[0262] 2.4 Pharmaceutical Composition

[0263] In some embodiments, the present application provides a pharmaceutical composition comprising a genetically modified eukaryotic cell of the present application or a population of genetically modified eukaryotic cells of the present application, and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be prepared according to known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (21st ed., Philadelphia, Lippincott, Williams & Wilkins, 2005). In making the pharmaceutical preparations according to the present application, the cells are typically combined with a pharmaceutically acceptable carrier and the resulting composition is administered to a subject. Of course, the carrier must be acceptable in the sense of being compatible with any other ingredients in the formulation and not injurious to the subject. In some embodiments, the pharmaceutical compositions of the present application can further comprise one or more additional agents useful in treating a disease in a subject. In additional embodiments, the pharmaceutical compositions of the present application can further comprise a biological molecule, such as a cytokine (e.g., IL-2, IL-7, IL-15, and / or IL-21), that promotes cell proliferation and engraftment of genetically modified T cells in vivo. The pharmaceutical compositions comprising genetically modified eukaryotic cells of the present application can be administered with the additional agents or biological molecules in the same composition, or can be co-administered in separate compositions.

[0264] The present disclosure also provides a genetically modified cell or population thereof described herein for use as a medicament. The present disclosure further provides use of a genetically modified cell or population thereof described herein in the manufacture of a medicament for treating a disease in a subject in need thereof. In one such aspect, the medicament can be for cancer immunotherapy in a subject in need thereof.

[0265] It is known that cells in which the presently disclosed meganucleases have been introduced can reduce off-target cleavage, reduce the persistence of the meganuclease in the cell, increase the efficiency of disruption of the TCR alpha constant region gene, enhance (i.e., increase) CAR T expansion, and have lower differentiation compared to cells that have been genetically modified with the TRC1-2x.87EE meganuclease, in some embodiments, the presently disclosed pharmaceutical compositions comprising genetically modified cells also have improved efficacy in treating a disease (e.g., cancer) when administered to a subject in need thereof compared to administration of a pharmaceutical composition comprising cells genetically modified with the TRC1-2x.87EE meganuclease.

[0266] In some embodiments, the presently disclosed engineered meganucleases, when introduced into a population of cells, produce a higher percentage of a population of TCR- and CAR+ cells compared to the first generation TCR 1-2x.87EE meganuclease when introduced into a population of cells.

[0267] In addition, cells that have been genetically modified with the presently disclosed engineered meganucleases exhibit improved properties compared to cells that have been genetically modified with the TRC1-2x.87EE meganuclease, including reduced off-target cleavage and effects thereof, reduced persistence of the meganuclease in the cell, enhanced (i.e., increased) CAR T expansion, and lower cell differentiation. In addition, a population of cells in which the presently disclosed meganuclease (or nucleic acid encoding the same) has been introduced has a greater percentage of modified cells and a greater percentage of less differentiated cells compared to a population of cells in which the TRC1-2x.87EE meganuclease (or nucleic acid encoding the same) has been introduced. In particular embodiments, a population of cells in which the presently disclosed engineered meganuclease has been introduced exhibits a greater percentage of central memory T cells (e.g., those expressing CD45RO, CCR7, and CD62L) compared to a population of cells in which the parental TRC1-2x.87EE meganuclease has been introduced.

[0268] The pharmaceutical compositions of the present application can be used to treat any disease state that can be targeted by T cell adoptive immunotherapy. In particular embodiments, the pharmaceutical compositions and medicaments of the present application can be used to treat cancer. Non-limiting examples of cancers that can be treated with the pharmaceutical compositions and medicaments of the present disclosure are carcinomas of the epithelium, lymphomas, sarcomas, melanomas, blastomas, leukemias, and germ cell tumors, including but not limited to cancers of B-cell origin, neuroblastoma, osteosarcoma, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, hepatocarcinoma, gastric carcinoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, cancer of the stomach, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers (including those induced by asbestos), multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia, immunoblastic large cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, and T-cell lymphoma, and any combination of said cancers. In certain embodiments, cancers of B-cell origin include, but are not limited to, B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell lymphoma, diffuse large B-cell lymphoma, pre-B ALL (pediatric indication), mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Burkitt's lymphoma, multiple myeloma, and B-cell non-Hodgkin's lymphoma.

[0269] In some of these embodiments in which a cancer is treated with the presently disclosed genetically modified cells or populations thereof, the subject to whom the genetically modified cells or populations thereof are administered is further administered an additional therapeutic agent, such as radiation, surgery, or a chemotherapeutic agent.

[0270] The present application further provides populations of genetically modified cells comprising a plurality of the genetically modified cells described herein, which comprise in their genome an exogenous nucleic acid molecule encoding a sequence of interest, wherein the exogenous nucleic acid molecule is inserted into a T cell receptor alpha constant gene, and wherein cell surface expression of an endogenous TCR is reduced. Thus, in various embodiments of the application, there is provided a population of genetically modified cells, wherein at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of the cells in the population are genetically modified cells described herein. In other embodiments of the application, there is provided a population of genetically modified cells, wherein at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of the cells in the population are genetically modified cells described herein that also express a chimeric antigen receptor.

[0271] 2.5. Methods of administering genetically modified cells

[0272] Another aspect disclosed herein is the administration of an effective amount of the genetically modified eukaryotic cells of the present disclosure, or a population thereof, to a subject in need thereof. In particular embodiments, the pharmaceutical compositions described herein are administered to a subject in need thereof. For example, an effective amount of a population of cells can be administered to a subject having a disease. In particular embodiments, the disease can be cancer, and administration of the genetically modified eukaryotic cells of the present application represents an immunotherapy. The administered cells are capable of reducing the proliferation, reducing the number, or killing target cells in the recipient. Unlike antibody therapies, the genetically modified eukaryotic cells of the present disclosure are capable of replicating and expanding in vivo, leading to long-term persistence, which can result in sustained control of the disease.

[0273] Examples of possible routes of administration include parenteral (e.g., intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), or infusion). In addition, the administration can be by continuous infusion or by single or multiple boluses. In particular embodiments, the agent is infused over a period of less than about 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour. In other embodiments, the infusion occurs slowly at first and then increases over time.

[0274] In some embodiments, the genetically modified eukaryotic cells or populations thereof of the present disclosure target a tumor antigen for the purpose of treating a cancer. Such cancers can include, but are not limited to, an epithelial cancer, a lymphoma, a sarcoma, a blastoma, a leukemia, a cancer of B-cell origin, a breast cancer, a gastric cancer, a neuroblastoma, an osteosarcoma, a lung cancer, a melanoma, a prostate cancer, a colon cancer, a renal cell carcinoma, an ovarian cancer, a rhabdomyosarcoma, a leukemia, and a Hodgkin lymphoma. In particular embodiments, the cancers and disorders include, but are not limited to, pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, salvage after allogeneic bone marrow transplantation, etc. These cancers can be treated using a combination of CARs targeting, for example, CD19, CD20, CD22, and / or ROR1. In some non-limiting examples, the genetically modified eukaryotic cells or populations thereof of the present disclosure target an epithelial cancer, a lymphoma, a sarcoma, a melanoma, a blastoma, a leukemia, and a germ cell tumor, including, but not limited to, a cancer of B-cell origin, a neuroblastoma, an osteosarcoma, a prostate cancer, a renal cell carcinoma, a rhabdomyosarcoma, a liver cancer, a stomach cancer, a bone cancer, a pancreatic cancer, a skin cancer, a head and neck cancer, a breast cancer, a lung cancer, a cutaneous or intraocular malignant melanoma, a kidney cancer, a uterine cancer, an ovarian cancer, a colorectal cancer, a colon cancer, a rectal cancer, a cancer of the anal region, a cancer of the stomach, a testicular cancer, a uterine cancer, a carcinoma of the fallopian tubes, a carcinoma of the endometrium, a carcinoma of the cervix, a carcinoma of the vagina, a carcinoma of the vulva, a non-Hodgkin lymphoma, an esophageal cancer, a small bowel cancer, a cancer of the endocrine system, a cancer of the thyroid gland, a cancer of the parathyroid gland, a cancer of the adrenal gland, a soft tissue sarcoma, a cancer of the urethra, a cancer of the penis, a childhood solid tumor, a lymphocytic lymphoma, a bladder cancer, a cancer of the kidney or ureter, a carcinoma of the renal pelvis, a neoplasm of the central nervous system (CNS), a primary CNS lymphoma, a tumor angiogenesis, a spinal axis tumor, a brain stem glioma, a pituitary adenoma, Kaposi's sarcoma, an epidermoid cancer, a squamous cell cancer, an environmentally induced cancer (including a cancer induced by asbestos), a multiple myeloma, a Hodgkin lymphoma, a non-Hodgkin lymphoma, an acute myelogenous lymphoma, a chronic myelocytic leukemia, a chronic lymphocytic leukemia, an immunoblastic large cell lymphoma, an acute lymphoblastic leukemia, a mycosis fungoides, an anaplastic large cell lymphoma, and a T-cell lymphoma, and any combination of said cancers. In certain embodiments, the cancer of B-cell origin includes, but is not limited to, a B-lineage acute lymphoblastic leukemia, a B-cell chronic lymphocytic leukemia, a B-cell lymphoma, a diffuse large B-cell lymphoma, a pre-B ALL (pediatric indication), a mantle cell lymphoma, a follicular lymphoma, a marginal zone lymphoma, a Burkitt lymphoma, a multiple myeloma, and a B-cell non-Hodgkin lymphoma.

[0275] When an "effective amount" or "therapeutic amount" is indicated, it can be determined by a physician, in accordance with the individual needs of the patient (subject), taking into account the age, body weight, size of tumor if present, extent of infection or metastasis, and the condition of the patient (subject). In some embodiments, a pharmaceutical composition comprising the genetically modified cells described herein or populations thereof is administered at a dose of 10 4 to 10 9 cells / kg body weight, including all integer values within those ranges. In further embodiments, the dose is 10 5 to 10 6 cells / kg body weight, including all integer values within those ranges. In some embodiments, the cell composition is administered multiple times at these doses. The cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0276] In some embodiments, administration of the genetically modified eukaryotic cells of the present disclosure or populations thereof alleviates at least one symptom of the target disease or disorder. For example, administration of the genetically modified T cells of the present disclosure or populations thereof can alleviate at least one symptom of a cancer. Symptoms of cancer are well known in the art and can be determined by known techniques.

[0277] 2.6 Methods for generating recombinant viral vectors

[0278] In some embodiments, the present application provides viral vectors (e.g., recombinant AAV vectors) for use in the methods of the present application. Recombinant AAV vectors are typically produced in mammalian cell lines such as HEK-293. Because the viral cap and rep genes are removed from the vector to prevent self-replication, making room for the therapeutic gene (e.g., meganuclease gene) to be delivered, it is necessary to provide these genes in trans in the packaging cell line. In addition, it is necessary to provide the "helper" (e.g., adenovirus) components necessary to support replication (Cots et al. (2013), Curr. Gene Ther. 13(5):370-81). Typically, recombinant AAV vectors are produced using triple transfection, in which a cell line is transfected with a first plasmid encoding the "helper" components, a second plasmid containing the cap and rep genes, and a third plasmid containing the viral ITRs, which contain the intervening DNA sequences to be packaged into the virus. The viral particles containing the genome (ITRs and intervening gene of interest) encased in the capsid are then isolated from the cells by freeze-thaw cycles, sonication, detergents, or other means known in the art. The particles are then purified using cesium chloride density gradient centrifugation or affinity chromatography, and the gene of interest is then delivered to a cell, tissue, or organism (e.g., a human patient).

[0279] Because recombinant AAV particles are typically produced (manufactured) in cells, precautions must be taken to ensure that the engineered meganuclease is not expressed in the packaging cell when practicing the present application. Because the viral genome of the present application can contain recognition sequences for the meganuclease, any meganuclease expressed in the packaging cell line can be able to cleave the viral genome before it is packaged into the viral particle. This would result in reduced packaging efficiency and / or packaging of fragmented genomes. Several methods can be used to prevent expression of the meganuclease in the packaging cell, including:

[0280] The meganucleases can be placed under the control of tissue-specific promoters that are inactive in the packaging cells. For example, if the viral vector is developed to deliver one or more meganuclease genes to muscle tissue, a muscle-specific promoter can be used. Examples of muscle-specific promoters include C5-12 (Liu et al. (2004) Hum Gene Ther. 15:783-92), the muscle-specific creatine kinase (MCK) promoter (Yuasa et al. (2002) Gene Ther. 9: 1576-88), or the smooth muscle 22 (SM22) promoter (Haase et al. (2013) BMC Biotechnol. 13:49-54). Examples of CNS (neuronal) specific promoters include the NSE, synapsin, and MeCP2 promoters (Lentz et al. (2012) Neurobiol Dis. 48: 179-88). Examples of liver-specific promoters include the albumin promoter (e.g., Palb), human alpha 1 -antitrypsin (e.g., PalAT), and hemopexin (e.g., Phpx) (Kramer et al., (2003) Mol. Therapy 7:375-85), the hybrid liver-specific promoter (liver locus control region from the ApoE gene (ApoE-HCR) and the liver-specific alpha 1 -antitrypsin promoter), the human transthyretin binding globulin (TBG) promoter, and the apolipoprotein A-II promoter. Examples of eye-specific promoters include the opsin and corneal epithelium-specific K12 promoter (Martin et al. (2002) Methods (28):267-75) (Tong et al., (2007) J Gene Med, 9:956-66). These promoters or other tissue-specific promoters known in the art are not highly active in HEK-293 cells, and thus, when incorporated into the viral vectors of the application, would not be expected to produce significant levels of meganuclease gene expression in the packaging cells. Similarly, the viral vectors of the application contemplate the use of other cell lines with tissue-specific promoters that are not compatible (i.e., the well-known HeLa cell line (human epithelial cells) with the liver-specific hemopexin promoter). Other examples of tissue-specific promoters include: synovial sarcoma PDZD4 (cerebellum), C6 (liver), ASB5 (muscle), PPP1R12B (heart), SLC5A12 (kidney), cholesterol-regulated APOM (liver), ADPRHL1 (heart), and monogenic malformation syndrome TP73L (muscle). (Jacox et al., (2010), PLoS One v. 5(8): e12274).

[0281] Alternatively, the vector can be packaged in cells from a different species that is unlikely to express the meganuclease. For example, viral particles can be produced in microbial, insect, or plant cells using a mammalian promoter that is not active in the non-mammalian packaging cells, such as the well-known cytomegalovirus or SV40 virus early promoters. In a preferred embodiment, viral particles are produced in insect cells using a baculovirus system as described by Gao et al. (Gao et al. (2007), J. Biotechnol. 131(2): 138-43). The meganuclease under the control of a mammalian promoter is unlikely to be expressed in these cells (Airenne et al. (2013), Mol. Ther. 21(4): 739-49). Moreover, insect cells utilize different mRNA splicing motifs than mammalian cells. Thus, it is possible to incorporate a mammalian intron, such as the human growth hormone (HGH) intron or the SV40 large T antigen intron, into the coding sequence of the meganuclease. Because these introns cannot be efficiently spliced from the pre-mRNA transcript in insect cells, the insect cells will not express a functional meganuclease and package the full-length genome. In contrast, the mammalian cells to which the resulting recombinant AAV particles are delivered will properly splice the pre-mRNA and express a functional meganuclease protein. Haifeng Chen reported the use of HGH and SV40 large T antigen introns to attenuate expression of the toxic proteins Bacillus cerein and diphtheria toxin fragment A in insect packaging cells, enabling the production of recombinant AAV vectors carrying these toxin genes (Chen (2012), Mol Ther Nucleic Acids. 1(11): e57).

[0282] The meganuclease gene can be operably linked to an inducible promoter, such that meganuclease expression requires a small molecule inducer. Examples of inducible promoters include the Tet-On system (Clontech; Chen et al. (2015), BMC Biotechnol. 15(1):4) and the RheoSwitch system (Intrexon; Sowa et al. (2011), Spine, 36(10):E623-8). Both systems, as well as similar systems known in the art, rely on ligand-induced transcription factors (Tet repressor and variants of the ecdysone receptor, respectively) that activate transcription in response to small molecule activators (doxycycline or ecdysone, respectively). Implementing the application using such ligand-inducible transcriptional activators involves: 1) placing the meganuclease gene under the control of a promoter responsive to the corresponding transcription factor, the meganuclease gene having one or more binding sites for the transcription factor; and 2) including in the packaged viral genome a gene encoding the transcription factor. The latter step is necessary because, if the transcriptional activator is not provided to the target cell at the same time, the meganuclease will not be expressed in the target cell or tissue after delivery of the recombinant AAV. The transcriptional activator then induces meganuclease gene expression only in cells or tissues treated with the cognate small molecule activator. This approach is advantageous because it enables spatiotemporal regulation of meganuclease gene expression by choosing when and to which tissues to deliver the small molecule inducer. However, the requirement to include the inducer in the viral genome, which greatly limits the carrying capacity, is a drawback of this approach.

[0283] In another preferred embodiment, recombinant AAV particles are produced in a mammalian cell line that express a transcriptional repressor that prevents expression of the meganuclease. Transcriptional repressors are known in the art and include Tet repressor, Lac repressor, Cro repressor, and Lambda repressor. Many nuclear hormone receptors, such as ecdysone receptors, can also act as transcriptional repressors in the absence of cognate hormone ligand. To practice the present application, the packaging cells are transfected / transduced with a vector encoding the transcriptional repressor, and the meganuclease gene in the viral genome (packaging vector) is operably linked to a promoter modified to contain a repressor binding site, such that the repressor silences the promoter. The gene encoding the transcriptional repressor can be placed in multiple locations. It can be encoded on a separate vector; it can be incorporated into the packaging vector outside the ITR sequences; it can be incorporated into the cap / rep vector or adenovirus helper vector; or it can be stably integrated into the genome of the packaging cell, such that it is constitutively expressed. Methods of modifying common mammalian promoters to incorporate transcriptional repressor sites are known in the art. For example, Chang and Roninson modified the strong constitutive CMV and RSV promoters to contain an operator for the Lac repressor, and showed that gene expression from the modified promoters was greatly attenuated in cells expressing the repressor (Chang and Roninson (1996), Gene 183: 137-42). The use of a non-human transcriptional repressor ensures that transcription of the meganuclease gene is inhibited only in the packaging cells expressing the repressor, and not in the target cells or tissues transduced with the resulting recombinant AAV vectors.

[0284] 2.7 Engineered nuclease variants

[0285] Embodiments of the present application include the engineered nucleases described herein and variants thereof. Other embodiments of the present application include polynucleotides comprising nucleic acid sequences encoding the nucleases described herein, as well as variants of such polynucleotides.

[0286] As used herein, "variant" is intended to mean a substantially similar sequence. A "variant" polypeptide means a polypeptide derived from a "native" polypeptide by deletion or addition of one or more amino acids at one or more internal sites of the native protein and / or substitution of one or more amino acids at one or more sites in the native polypeptide. As used herein, a "native" polynucleotide or polypeptide includes the parent sequence from which a variant is derived. Variant polypeptides encompassed by embodiments have biological activity. That is, they continue to possess the biological activity required of the native protein; i.e., the ability to recognize and cleave the TRC 1-2 recognition sequence (SEQ ID NO: 5) found in the human T cell receptor alpha constant region (SEQ ID NO: 3), and in some embodiments, exhibit at least one improved property selected from the following relative to the first generation TRC 1-2 meganuclease: improved (i.e., increased) specificity and off-target cleavage, reduced intracellular persistence, and enhanced (i.e., increased) efficiency of modification of the TCR alpha constant region gene. Such variants can be produced, for example, by human manipulation. Biological activity variants of the native polypeptides of embodiments (e.g., SEQ ID NOs: 7 and 8), or of the recognition half-site binding subunits described herein, will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92% about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the amino acid sequence of the native polypeptide or native subunit, as determined by sequence alignment programs and parameters described elsewhere herein. Biological activity variants of the polypeptides or subunits of embodiments can differ from that polypeptide or subunit by as few as about 1-40 amino acid residues, as few as about 1-20, as few as about 1-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acid residues.

[0287] The polypeptides of the embodiments can be altered in various ways including amino acid substitutions, deletions, truncations and insertions. Methods for such manipulations are known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alteration are well known in the art. See, e.g., 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; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and references cited therein. Guidance in determining which amino acid substitutions are likely to produce a biologically active protein can be found in Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), which is incorporated herein by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, can be optimal.

[0288] In some embodiments, the engineered meganucleases of the application can comprise variants of the HVR1 and HVR2 regions disclosed herein. The parent HVR region can comprise, for example, residues 24-79 or residues 215-270 of the exemplified engineered meganucleases. Thus, the variant HVR can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the amino acid sequence corresponding to residues 24-79 or residues 215-270 of the engineered meganucleases exemplified herein, such that the variant HVR region retains the biological activity of the engineered meganuclease (i.e., binds to and cleaves the recognition sequence). Furthermore, in some embodiments of the application, the variant HVR1 region or the variant HVR2 region can comprise a residue corresponding to an amino acid residue present at a particular position within the parent HVR. In this context, "corresponding to" means that the amino acid residue in the variant HVR is the same amino acid residue (i.e., the same residue alone) present in the parent HVR sequence at the same relative position (i.e., relative to the rest of the amino acids of the parent sequence). For example, if the parent HVR sequence comprises a serine residue at position 26, then a variant HVR comprising a residue "corresponding to" residue 26 would also comprise a serine at the position relative to (i.e., corresponding to) the parent position 26.

[0289] In particular embodiments, the engineered meganucleases of the application comprise an HVR1 region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7.

[0290] In certain embodiments, the engineered meganucleases of the application comprise an HVR2 region having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7.

[0291] In some embodiments, the engineered meganuclease of the application comprises a HVR2 region having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 8.

[0292] In particular embodiments, the engineered meganuclease of the application comprises a HVR1 region having at least 97% sequence identity to the amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7 and a HVR2 region having at least 81 % sequence identity to the amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7.

[0293] In other particular embodiments, the engineered meganuclease of the application comprises a HVR1 region having the amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 8 and a HVR2 region having at least 86% sequence identity to the amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 8.

[0294] A large number of amino acid modifications to the DNA recognition domain of wild-type I-CreI meganucleases have been previously identified (e.g. US 8,021,867), which individually or in combination, result in recombined meganucleases having altered specificity at a single base within the recognition sequence half-site, such that the resulting rationally designed meganucleases have different half-site specificities from the wild-type enzyme. Table 2 provides potential substitutions that can be made in the engineered meganuclease monomer or subunit based on the presence of the base at each half-site position (-1 to -9) of the recognition half-site to enhance specificity.

[0295] Table 2.

[0296]

[0297]

[0298] Bold are wild-type contact residues and do not constitute "modifications" as used herein. Asterisks indicate that the residue contacts the base on the antisense strand.

[0299] Certain modifications can be made in the engineered meganuclease monomers or subunits to modulate DNA binding affinity and / or activity. For example, the engineered meganuclease monomers or subunits described herein can comprise a G, S, or A at the residue corresponding to position 19 of I-Crel or SEQ ID NO: 7 or 8 (WO 2009001159), a Y, R, K, or D at the residue corresponding to position 66 of I-Crel or SEQ ID NO: 7 or 8, and / or an E, Q, or K at the residue corresponding to position 80 of I-Crel or SEQ ID NO: 7 or 8 (US8021867).

[0300] For a polynucleotide, a "variant" includes the deletion and / or addition of one or more nucleotides at one or more sites within the native polynucleotide. Those of skill in the art will recognize that variants of the nucleic acids of the embodiments are constructed such that the open reading frame is maintained. For a polynucleotide, conservative variants include those sequences that, due to the degeneracy of the genetic code, encode an amino acid sequence of one of the polypeptides of the embodiments. Variant polynucleotides include synthetically derived polynucleotides, such as those generated, for example, by the use of site-directed mutagenesis, but which still encode a recombinant nuclease of the embodiments. Generally, a variant of a particular polynucleotide of the embodiments will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to that particular polynucleotide, as determined by sequence alignment programs and parameters described elsewhere herein. Variants of a particular polynucleotide of the embodiments (i.e., the reference polynucleotide) can also be assessed by comparing the percent sequence identity between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide.

[0301] It is contemplated that deletions, insertions and substitutions of variant protein sequences encompassed herein will not produce a profound change in the properties of the polypeptide. However, when such changes could alter the reading frame, the skilled artisan will appreciate that the effect is not a substitution mutation, but rather a frame shift mutation which will significantly alter the polypeptide encoded by a nucleotide sequence. When it is difficult to predict the exact effect of a substitution, deletion or insertion, the skilled artisan will appreciate that the effect can be evaluated by routine screening tests, for example, by the ability of the polypeptide to preferentially recognize and cleave the TRC 1-2 recognition sequence (SEQ ID NO: 5) found within exon 1 of the human T cell receptor alpha constant region gene (SEQ ID NO: 3).

[0302] Example

[0303] The application is further illustrated by the following examples which should not be construed as limiting. Using only routine experimentation, one of ordinary skill in the art will recognize or be able to ascertain many equivalents to the specific substances and procedures described herein. Such equivalents are intended to be encompassed by the following claims.

[0304] Example 1

[0305] Characterization of a wide range of nucleases specific for the TRC 1-2 recognition sequence

[0306] 1. A wide range of nucleases that recognize and cleave TRC 1-2 recognition sequences.

[0307] Second generation TRC 1-2 meganucleases, designated TRC 1-2L.1592 (SEQ ID NO: 7) and TRC 1-2L.1775 (SEQ ID NO: 8), were engineered to recognize and cleave the TRC 1-2 recognition sequence (SEQ ID NO: 5) found in the human T cell receptor alpha constant region. Each of these second generation meganucleases comprises an N-terminal nuclease localizing signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit in each TRC 1-2 meganuclease binds to the TRC1 recognition half-site of SEQ ID NO: 5, while the second subunit binds to the TRC2 recognition half-site (see Figure 1 ). The TRC1 -binding subunit and the TRC2-binding subunit each comprise a 56 base pair hypervariable region, designated HVR1 and HVR2, respectively.

[0308] The HVR1 region of each TRC1 -binding subunit consists of residues 215-270 of SEQ ID NO: 7 and 8. The TRC1 -binding subunits of TRC 1-2L.1592 and TRC 1-2L.1775 are identical to each other outside of the HVR1 region. The HVR1 region of each TRC 1-2 meganuclease comprises modifications at positions 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 266, and 268 relative to the wild-type I-CreI sequence (SEQ ID NO: 1). Although not modified relative to wild-type I-CreI, the arginine residue at position 261 of SEQ ID NO: 7 and 8 is believed to contribute to the specificity of the nuclease along with the modified HVR1 residues. The HVR1 region of TRC 1-2L.1592 shares 96.43% sequence identity with the HVR1 region of the TRC 1-2x.87EE meganuclease. The HVR1 region of TRC 1-2L.1775 shares 100% sequence identity with the HVR1 region of the TRC 1-2x.87EE meganuclease.

[0309] The HVR2 region of each TRC2 binding subunit consists of residues 24-79 of SEQ ID NOS: 7 and 8. The TRC2 binding subunits of TRC 1-2L.1592 and TRC 1-2L.1775 are identical to each other outside of the HVR2 region, except for position 80 of SEQ ID NOS: 7 and 8, which can be E (TRC 1-2L.1592) or Q (TRC 1-2L.1775). The HVR2 region of each TRC 1-2 meganuclease comprises modifications at positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 48, 50, 68, 70, 75, and 77 relative to the wild-type I-CreI sequence (SEQ ID NO: 1). The TRC 1-2L.1592 meganuclease also comprises modifications at positions 71, 72, and 73 relative to wild-type I-CreI. Also of note is that the arginine residue at position 139 of SEQ ID NOS: 7 and 8 is modified relative to the wild-type I-CreI sequence and is believed to contribute to the specificity of the nuclease along with the modified HVR2 residues. The HVR2 region of TRC 1-2L.1592 shares only 80.36% sequence identity with the HVR2 region of the first generation TRC 1-2x.87EE meganuclease. The HVR2 region of TRC 1-2L.1775 shares only 85.71% sequence identity with the HVR2 region of the TRC 1-2x.87EE meganuclease.

[0310] 2. First Generation TRC Optimization of 1-2 nucleases

[0311] The recognition site specificity of the previously reported TRC 1-2x.87EE meganuclease was assessed using a method very similar to that of GUIDE-seq (Tsai et al. (2015), Nat Biotechnology 33: 187-197), but adapted to find potential off-target sites of the meganuclease. In general, potential off-target sites are identified by capturing a probe oligonucleotide in a double-stranded DNA break. The TRC 1-2 meganucleases generate a 4 base pair 3’ overhang, so the probe oligonucleotide also comprises a randomized four base pair overhang to improve the efficiency of ligation at sites more likely to be generated by nuclease cleavage.

[0312] Specificity analysis of TRC 1-2x.87EE found multiple potential off-target sites in human T cells. These off-targets can be divided into two relevant categories: unique targets that are hit with high frequency and repetitive targets that are hit with low frequency. The key amino acids involved in the recognition of these off-targets were re-randomized. Subsequently, a simultaneous selection for cleavage of the intended site and counter-selection for off-target sites that do not cleave was performed. The off-target sites were alternated between successive selection rounds to isolate the answer that distinguishes between the two off-targets (i.e. the nuclease). The two off-targets used were Offl : 5'-TGGCCTGGAGaAACAgtgtaaa-3' (SEQ ID NO: 16), which is a low frequency cleaving but highly repetitive site in the genome; and Off2: 5'-cGGCCTGtAGtAcaggAcCTGA-3' (SEQ ID NO: 17), which is a frequently hit, unique off-target (lowercase letters indicate mismatches to the intended site). A variety of nuclease libraries were used.

[0313] After selection, a 96-well plate of isolated clones from each successful library was prepared to isolate plasmid DNA. Each plasmid DNA was individually transfected into CHO cells containing an integration target site in an interruption between two direct repeat sequences in the GFP gene. Cleavage of the target site results in repair of the GFP gene through single-strand annealing, and the frequency of cleavage of the target site can be counted by counting the number of GFP positive cells on a flow cytometer. We analyzed the nuclease plasmids against cells with the intended site and the Offl target site. In this way, we can assess which nucleases still cleave the intended site, but best distinguish against the off-target. We identified five candidates. Three of the candidates, L.1462, L.1466, and L.1469, were re-isolated from the original library of TRC 1-2. These three answers were all unique, but related to each other. Two of the candidates, L.1108 and L.1118, were isolated from TRC library 2. Each of these candidates represents an intermediate nuclease in the development of the second generation of nucleases of the invention.

[0314] To further improve the nucleases, the key amino acids involved in recognition site specificity were randomized. L.1462, L.1466, and L.1469 were pooled into one library, and L.1108 and L.1118 were pooled into a second library. New randomizations were introduced in both by PCR. The new libraries employed a similar selection strategy; selections were performed simultaneously for the intended site and against Offl or Off2. Off-targets were alternated between selection rounds. The 96-well plates yielding individual answers from the selection were tested in the CHO iGFFP assay to determine cleavage of the intended site as well as both Offl and Off2. Several new nucleases were identified from this additional round of optimization. Answers from the library based on L.1462, L.1466, and L.1469 included: L.1775 and L.1843. One answer from the library based on L.1108 and L.1118: L.1592. All new nucleases showed strong activity on the intended target and strong discrimination against both off-targets (described below). The new nucleases were subjected to an oligonucleotide capture assay (further described below) to determine potential off-target sites and demonstrated a reduction in the number of potential off-targets overall and, in particular, L.1592 had very few potential rational off-target sites. L.1108, L.1469, L.1592, L.1775, and L.1843 were subjected to a further 7-day evaluation in the iGFFP assay to measure the stability of the GFP signal over time, a general measure of toxicity. L.1469, L.1592, L.1775, and L.1843 were further tested for function in primary T cells.

[0315] 3. Evaluation of TRC 1-2 identification sequence cutting and off-target cutting

[0316] To determine whether the TRC 1-2 meganucleases could recognize and cleave the TRC 1-2 recognition sequence (SEQ ID NO: 5), each TRC 1-2 meganuclease was evaluated using the previously described CHO cell reporter assay (see WO / 2012 / 167192, Figure 3 ) To perform this analysis, a pair of CHO cell reporter lines were prepared that carry a non-functional green fluorescent protein (GFP) gene expression cassette integrated into the cell genome. The GFP gene in each cell line is interrupted by a pair of recognition sequences, such that intracellular cleavage of either recognition sequence by a meganuclease stimulates a homologous recombination event that results in a functional GFP gene. In both cell lines, one of the recognition sequences is derived from the TRC 1-2 gene, and the second recognition sequence is specific for a control meganuclease known as "CHO 23 / 24". CHO reporter cells containing the TRC 1-2 recognition sequence (SEQ ID NO: 5) and the CHO 23 / 24 recognition sequence are referred to herein as "TRC 1-2 cells".

[0317] TRC 1-2 cells were transfected with plasmid DNA encoding one of the TRC 1-2 meganucleases (e.g. TRC 1-2 x.87EE, TRC 1-2 L.1592, TRC 1-2 L.1775 or TRC 1-2 L.1843) or encoding the CHO 23 / 34 meganuclease. 4e5 CHO cells were transfected with 50 ng of plasmid DNA in a 96 well plate using Lipofectamine 2000 (ThermoFisher) according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were assessed by flow cytometry to determine the percentage of GFP positive cells compared to untransfected negative controls (1-2bs). It was found that all TRC 1-2 meganucleases produced GFP positive cells in the cell line containing the TRC 1-2 recognition sequence at a frequency that was significantly above the negative control and comparable to or above the CHO 23 / 24 positive control, indicating that each TRC 1-2 meganuclease was able to effectively recognize and cleave the intended TRC 1-2 recognition sequence in the cell Figure 4 A-4C).

[0318] Alternatively, TRC 1-2 meganucleases were also transfected into TRC Offl and TRC Off2 cells, which contain an inversely selected off-target sequence between the GFP direct repeat sequences. Unlike the intended target site TRC 1-2 CHO cells, the desired nuclease in the TRC Offl and TRC Off2 CHO cells had only background levels of GFP positive cells because it was able to discriminate against cleaving the off-target sequence. In these experiments, the CHO 23-24 target site served as a positive control, showing that GFP could still be produced if the CHO 23-24 nuclease cleaved the target site. The new nucleases showed a significant improvement (i.e. increase) in discrimination against the Offl and Off2 target sites compared to TRC 1-2 x.87EE, with levels of %GFP comparable to the TRC 1-2bs negative control Figure 5 A-5C).

[0319] The efficacy of the TRC 1-2. L1469, L.1592, L.1775 and L.1843 engineered meganucleases was also determined in a time dependent manner at 2, 5 and 7 days after introducing the meganuclease mRNA into TRC 1-2 cells. In this study, TRC 1-2 cells (1.0 x 10 6 ) were transfected with 1 x 10 6Copy L.1469 mRNA electroporation. 48 hours post transfection, cells were assessed by flow cytometry to determine the percentage of GFP positive cells. CHO 23-24 meganuclease was also included as a positive control at each time point. Each meganuclease showed comparable percentage of GFP positive cells relative to CHO 23-24 Figure 6 and Figure 7 ). Only L.1469 showed a trend of decreasing GFP positive cells over time, indicating it had some unresolved toxicity issues that were improved in subsequent optimizations. The rest of the meganucleases showed stable or increasing GFP positive cells over time, at levels equal to or higher than the CHO 23-24 control.

[0320] The extended iGFP assay was also used to assess the discrimination of the same set of meganucleases against two off-targets, Offl and Off2, over a period of 7 days. In this case, cells containing Offl or Off2, as well as CHO 23-24, were electroporated with 1 x 10 6 Copy L.1469 mRNA electroporation. 48 hours post transfection, cells were assessed by flow cytometry to determine the percentage of GFP positive cells. CHO 23-24 meganuclease was also included as a positive control at each time point. Each meganuclease showed comparable percentage of GFP positive cells relative to CHO 23-24 Figure 8 A and 8B). L.1592 exhibited minimal cleavage of Offl or Off2, comparable to mock control cells. L.1469 showed some detectable cleavage of Offl and Off2, but it was much lower than the cleavage observed by TRC 1-2x.87EE. L.1775 and L.1843 showed improvement over their parent L.1469 in discrimination against off-targets.

[0321] 4. Oligonucleotide capture assay and off-target cleavage analysis

[0322] In these studies, the oligo capture assay was used to identify off-target cleavage induced by the TRC 1-2 meganuclease. Similar to GUIDE-seq, the oligo capture assay identifies potential off-target sites produced by the TRC 1-2 meganuclease by capturing oligonucleotides within the break site in cellular genomic DNA. GUIDE-seq was developed for DNA breaks produced by CRISPR-Cas9, and there were some key modifications to the chemistry and analysis in order to apply the technology to the current nuclease. Unlike CRISPR-cas9, the engineered meganucleases of the present invention produce a 4 base pair 3' overhang. To accommodate this difference, the oligonucleotides used in the oligo capture have a random four base pair overhang that can be compatible with the overhang produced by the TRC 1-2 meganuclease. Due to the higher efficiency of ligating sticky ends rather than blunt ends, a higher frequency of insertions was observed. Cells were transfected with mRNA encoding the nuclease and double stranded DNA oligonucleotides. Two days later, genomic DNA was isolated from these cells and sonicated to shear the DNA into smaller sizes. Oligonucleotide adaptors were ligated to the sheared DNA, and PCR was used to amplify any DNA fragments that contained the adaptor on one end and the captured oligonucleotide on the other end. The amplified DNA was purified and a sequencing library was prepared using standard commercial kits.

[0323] The sequencing library was run on an Illumina MiSeq using the V2 2xl50 kit. The data was filtered and the valid sites of the captured oligonucleotides were analyzed and potential off-target sites were predicted. Likewise, the experimental protocol required adjustments to the PAM search from that used for CRISPR-cas9 to the TRC 1-2 meganuclease search. Software developed checks each sequence to make sure that there is an adaptor and captured oligonucleotide flanking the sequence to verify that it is a valid read. The software also checks for PCR duplicates and removes identical reads to help reduce PCR bias. The sequence reads were aligned to the reference genome and the grouped sequences within a window of thousands of base pairs were scanned for potential TRC 1-2 meganuclease sites.

[0324] Each TRC 1-2 meganuclease is a linked dimer. Each monomer recognizes a nine base pair half-site with a four base pair spacer sequence in the center between the two half-sites. The software will find the closest sequence match for each half-site without allowing a gap. The intervening four base pairs are not considered in off-target selection because TRC 1-2 meganucleases can generally tolerate higher degeneracy at these positions of the target site. The software outputs a list of potential off-target sites with the number of base mismatches in the combined half-sites, but does not count mismatches in the intervening four base pairs. Unlike CRISPR-Cas9, which eliminates any off-targets identified with more than six mismatched base pairs, the software does not eliminate any off-targets based on an arbitrary mismatch filter. Instead, background noise arising from random capture of oligos at vulnerable or hot spots within the genome can be reduced in two ways. First, an untreated mock sample can also be run through the oligo capture, and the windows of integrated sites in the absence of nuclease can be subtracted from the nuclease-containing sample. We also found that running the assay in triplicate and eliminating any sites that are not repeated in at least two of the three replicates is a good way to empirically eliminate random integration noise.

[0325] While read counts do not directly correlate with the frequency of cleavage at a particular location, it is often possible to highlight off-targets that can be more interesting or more effective because they occur more frequently. Figure 9 One way to graphically visualize oligo capture data as a measure of the number of potential effective off-target sites is shown. Each off-target produced by a particular nuclease is plotted according to the number of unique sequence read counts of probe oligos captured at that site. The expected site should have the highest read count, which is the case for all of the TRC 1-2 meganucleases tested. Better nucleases remove higher count sites, and have fewer points above the background noise at the far left of the graph. Using this graph it is, for example, clear that TRC 1-2 L.1592 removes more of the higher read count sites than the first generation TRC 1-2 x.87EE.

[0326] Other visualization methods allow us to look at oligo capture data not only at the number of read counts recovered at a particular site, but also by the number of mismatches between the putative off-target site and the expected site. This allows a more accurate determination of true oligo integration sites compared to random integration or sequencing noise. In Figure 10In this case, off-target sites are plotted according to the number of aligned reads they have and the number of mismatched base pairs compared to the expected site is represented by color, with darker colors indicating a closer overall match between the off-target and the expected binding site. Boxes indicate regions of highest confidence. Off-targets within these boxes have high aligned read counts or very high similarity to the expected site, either of which reduces the likelihood that the site is background noise. Sites in the confidence regions are compared to the expected site, Figure 10 The specificity of the optimized meganucleases, especially TRC 1-2L.1592, was demonstrated to be improved compared to TRC 1-2x.87EE. TRC 1-2L.1592 showed a reduced number of sites with high read counts and a reduced number of sites with high similarity to the expected site.

[0327] Example 2

[0328] Optimized in vitro analysis of TRC 1-2 wide range of nucleases

[0329] 1. Assessment of gene editing efficiency, post-editing amplification, and differentiation.

[0330] In a first set of experiments, four optimized second-generation TRC 1-2 meganucleases were screened for their gene editing efficiency as well as their post-editing expansion and differentiation potential. Three different operators each evaluated all nuclease variants in T cells obtained from different healthy human T cell donors. The apheresis blood separation material was derived from donors K708, K799, and K6784 from Key Biologies (Memphis, TN). K708 and K6784 T cells were processed according to the following protocol: T cell enrichment using human CD3 positive selection reagent (StemCell Technologies), stimulation using ImmunoCult anti-CD2 / CD3 / CD28 (StemCell Technologies), and nuclease RNA delivery using 4D NucleoFEctor (Lonza). T cells from K799 were processed according to the following protocol: T cell enrichment using CD4 and CD8 microbeads and CliniMACS cell separator (Miltenyi Biotec), stimulation using TransAct (Miltenyi), and nuclease RNA delivery using MaxCyte-GT.

[0331] The editing efficiency, expansion and differentiation of four optimized nuclease variants (TRC 1-2L.1496, L.1592, L.1775 and L.1843) were compared against the progenitor nuclease TRC 1-2x.87EE and mock electroporated T cells. After three days of initial stimulation with ImmunoCult / TransAct, T cells were harvested, electroporated with RNA encoding one of the nucleases and then immediately transduced with an AAV6 vector encoding a CAR gene to be inserted into the TRC 1-2 cleavage site. Parallel control cultures that did not receive AAV were assembled.

[0332] On day 4 and day 8 post-editing, total cellularity was determined with a NucleoCounter NC-200 (ChemoMetec). Editing efficiency was determined by staining culture samples with antibodies against human CD3-PE (BioLegend clone UCHT1) and anti-FMC63 scFv-AlexaFluor647 (new clone produced in-house and conjugated). Differentiation was assessed by comparing the frequency of central memory, transitional memory and effector memory cells in the CD4 and CD8 compartments using CD4-BV786 (clone OKT4 BioLegend), CD8-BV711 (clone RPA-T8, BioLegend), CD62L-BB515 (clone SK11 BD Biosciences) and CD45RO-PE / Cy7 (clone UCHL1, BioLegend).

[0333] The results of these experiments are summarized in Figure 11 Knockout frequencies of endogenous T cell receptors (measured by T cells transitioning from CD3 positive to CD3 negative phenotype) were determined for each nuclease in 3 different donors. For all 3 donors tested (and using two cell preparation methods), both TRC 1-2L.1592 and L.1775 produced knockout cells at similar or higher efficiency than TRC 1-2x.87EE. In contrast, L.1469 and L.1843 produced lower knockout frequencies. This was true for all 3 donors tested. L.1775 exhibited slightly higher editing efficiency than L.1592. The increase in editing of the TRC 1-2 recognition sequence was associated with an increase in the rate of insertion of the CAR gene. In all three donors, L.1592 and L.1775 supported equivalent or superior editing and insertion frequencies.

[0334] Cell count data at day 8 post-edit was used to calculate CAR T cell fold expansion. In all three donors, L.1592, L.1775, and L.1843 promoted greater expansion than x.87EE following electroporation. In contrast, L.1469 promoted less expansion than x.87EE. In two of the three donors, L.1843 allowed the most extensive expansion of the three optimized nucleases. The degree of expansion supported by L.1775 varied from donor to donor.

[0335] CD4:CD8 ratio and memory subset data were also captured at day 8 post-edit. No major perturbations to the CD4:CD8 ratio were observed from any of the optimized nucleases compared to x.87EE, although L.1592, L.1775, and L.1843 generally resulted in a higher frequency of CD4+ cells. A greater degree of differentiation from central memory to transitional and effector memory populations was observed in cells edited with L.1469 compared to x.87EE. In contrast, equivalent or higher frequencies of cells maintained a central memory phenotype when edited with L.1592, L.1775, or L.1843.

[0336] These studies demonstrate that three of the four optimized nucleases outperformed TRC 1-2 x.87EE in terms of editing efficiency, cell expansion, and differentiation properties. One nuclease (L.1469) did not perform as well as x.87EE. Of the three variants with improved in vitro function, variant L.1775 supported the highest frequency of edited cells in culture, but supported the least amount of post-edit expansion and accelerated differentiation of T cells in culture. Variant L.1843 allowed the greatest amount of post-edit expansion and maintained a favorable central memory frequency, but was less efficient than L.1775 or L.1592 in terms of knockout frequency. Unexpectedly, L.1592 represented an improvement over the first generation x.87EE using all three criteria.

[0337] 2. Analysis of oligonucleotide capture assay and off-target cleavage

[0338] Oligonucleotide capture was performed on three replicates of T cells obtained from each of the three donors using the methods described previously in Example 1. Results of the oligonucleotide capture are shown in Figure 12. The dots represent the number of sequencing reads recovered at each putative off-target site as well as the expected target site. Putative sites with more than 7 mismatches to the expected target were removed because sites with more than 7 mismatches were not shown to be cleaved by TRC 1-2 L.1592 in previous studies. The expected target site for each sample is highlighted with a circle. The number of mismatches compared to the expected target is represented by the darkness of each circle, with fewer mismatches having darker colors. This figure represents oligonucleotide capture data, where the mock background was not removed, and read counts were normalized to the number of unique reads for each sample to account for differences in total number of reads recovered. As shown, TRC 1-2 L.1592 shows a small number of higher read count sites, as well as a small number of sites that are more similar to the expected site when compared to the sites used for editing and targeted insertion in the CAR T cell population.

[0339] 3. In vitro studies of editing efficiency, amplification, and cytokine secretion.

[0340] In the second set of in vitro studies, the efficiency of the second generation optimized TRC 1-2 meganuclease at editing T cells, the ability of edited T cells to expand after editing, and the ability of CAR T cells generated with the nuclease variant to respond to encounters with target cells bearing an antigen were assessed.

[0341] Apheresis blood separation material was sourced from donor K708 at Key Biologies (Memphis, TN) and T cells were enriched using human CD3 positive selection reagent (StemCell Technologies), stimulated using ImmunoCult anti-CD2 / CD3 / CD28 (StemCell Technologies), and nuclease RNA was delivered using a 4D NucleoFector (Lonza). Samples were run in triplicate in parallel.

[0342] The editing efficiency, expansion, and differentiation of the three optimized meganucleases (TRC 1-2 L.1592, L.1775, and L.1843) were compared to the progenitor cell nuclease TRC 1-2 x.87EE and mock electroporated T cells. Three days after initial stimulation with ImmunoCult / TransAct, T cells were harvested, electroporated with RNA encoding one of the nucleases, and then immediately transduced with an AAV6 vector encoding a CAR gene to be inserted into the TRC 1-2 cleavage site. On days 4 and 8 after editing, cultures were sampled to determine editing efficiency and expansion using a Beckman-Coulter CytoFLEX-LX flow cytometer. Endogenous T cell receptor knockout efficiency was assessed using anti-CD3-PE (BioLegend clone UCHT1), and CAR knockin was measured using anti-FMC62 scFv-AlexaFluor647 (new clone produced in-house and conjugated).

[0343] Proliferation, cytotoxicity, and cytokine production were assessed by co-culturing CAR T cells with CD19+ tumor lines Raji or Nalm6 at E:T ratios of 1 : 1 and 1 :2. CD19 negative K562 myeloid leukemia cells were used as a control. Culture supernatants were collected and analyzed for secreted cytokines using a Luminex MAGPIX instrument and a MilliPlex MAP 15-plex bead set (Millipore). Proliferation and targeted killing were assessed by staining culture cell samples with anti-CD4-APC (BioLegend clone OKT4), anti-CD8-FITC (BioLegend clone RPA-T8), and anti-CD19-PE (BioLegend clone HIB19), and using CytoFLEX-LX to acquire fluorescence data, as well as cell counts.

[0344] T cells edited with the TRC 1-2 meganucleases x.87EE and L.1775 had about 50% less total culture cellularity at day 8 compared to T cells without RNA electroporation (mock control) Figure 13 A). Cultures edited with L.1592 or L.1843 did not show total culture cellularity reduction to this extent. When considering editing efficiency, and calculating the total number of edited cells generated in the process, L.1592 generated the most TCR knockout cells Figure 13 B). Variants x.87EE and L.1775 generated nearly equal numbers of edited cells, while L.1843 generated the least. This pattern was also observed when measuring the number of CAR+ / TCR- cells in the cultures Figure 13 C).

[0345] CAR T cells produced with TRC 1-2x.87EE expanded near three-fold on the input number (defined by the horizontal dashed line) when the CAR T cells were co-cultured with target cells bearing the antigen Figure 14 ). Unexpectedly, CAR T cells produced with the optimized nucleases proliferated much more strongly than x.87EE, approaching a 10-fold expansion after 5 days. When the E:T ratio was increased to 1 :2, proliferation of x.87EE and L.1843 edited CAR T cells was reduced by about ½ relative to the 1 : 1 ratio. This was not observed with CAR T cells produced using L.1775 or L.1592, which were found to have significantly better performance (p<0.0001, Figure 15 A). When the number of remaining CD19+ Raji cells was measured (at an E:T ratio of 1 :2, Figure 15 B), all 4 CAR T products showed a 90% or greater reduction in Raji numbers compared to control cultures that did not receive CAR T cells. CAR T cells produced using the optimized nucleases eliminated Raji cells significantly better than cells produced using x.87EE.

[0346] Analysis of the co-culture supernatants showed that higher levels of effector cytokines were produced when CAR T cells were prepared using the optimized nucleases rather than x.87EE. L.1592 edited CAR T cells secreted the highest levels of IL-2, TNFa, IFNy, and granzyme B Figure 16 A-16D), and the second highest levels of perforin Figure 16 E). In the case of IL-2 and TNFa, the difference between cytokine production by x.87EE edited CAR T cells and L.1592 edited CAR T cells was 2-3 fold, while all other differences were small.

[0347] Overall, the optimized TRC 1-2 meganucleases L.1775, L.1592, and L.1843 are functionally superior to x.87EE. This is true in terms of the relative ability of the nucleases to support CAR T cell manufacture Figure 13 ), and the ability of the CAR T cells to respond to encounters with their target antigen Figure 14-16 ). From multiple experiments it can be concluded that while L.1775 generally supports the highest editing efficiency (knockout frequency), and L.1843 allows the greatest expansion of T cells after editing, L.1592 combines the second highest editing efficiency with the highest or second highest expansion to produce the highest overall number of CAR T cells. Importantly, CAR T cells produced with L.1592 show functional advantages (proliferation, target cell killing, and cytokine production) compared to the other optimized meganucleases.

[0348] 4. Optimized in vitro residence time of TRC 1-2 wide-range nucleases

[0349] Further studies were conducted to determine if the optimized second generation TRC 1-2 meganuclease had a shorter residence time in vitro than the first generation TRC 1-2x.87EE. A shorter residence time can be advantageous in the context of potential reductions in off-target cleavage and gene editing.

[0350] In these studies, T cells were obtained from apheresis blood separation products (Key Biologies) by magnetic enrichment of CD4+ and CD8+ cells using CD4 and CD8 microbeads and LD columns (Miltenyi). Cells were activated with anti-CD3 / anti-CD28 TransAct reagent (Miltenyi) for three days in Xuri media (GE) containing 5% FBS (GE Hyclone), 10 ng / ml IL-2 (Cellgenix), and 1% antibiotic / antimycotic solution (Gibco). Cells were then electroporated with in vitro transcribed mRNA (Trilink) encoding TRC 1-2x.87EE or TRC 1-2L.1592 nuclease using the MaxCyte electroporation system, 1 ug mRNA per 1e6 cells. Subsequently, cells were transduced with a recombining AAV6 vector carrying a donor template encoding an anti-CD19 chimeric antigen receptor designed for insertion at the TRC 1-2 site by homologous recombination (SAB Tech) in serum-free Xuri media containing 30 ng / ml IL-2 and 1% antibiotic / antimycotic solution. Six hours post-electroporation, samples were quantitated and resuspended in Xuri media containing 5% FBS, 30 ng / ml IL-2, and 1% antibiotic / antimycotic solution. At the 96 hour time point, residual unedited CD3+ T cells were removed from the TRC electroporated groups by magnetic depletion using LD columns, CliniMACS buffer, and CD3 microbeads (Miltenyi). Cells were then cultured in Xuri media + 5% FBS / 1% anti-anti + 10 ng / ml IL-15 and IL-21 at 37 degrees Celsius for the remainder of the experiment.

[0351] T cell samples were quantitated at 6 hours, 24 hours, 48 hours, 96 hours, and 168 hours post-electroporation, and equal amounts of viable cells were pelleted and resuspended in RIPA buffer (EMD Millipore) with protease inhibitors (Roche), mixed thoroughly, and either stored frozen or incubated on ice for 30 minutes before further processing as described below for Western blots.

[0352] Mock cells from the same donor were activated and cultured in the same media as the nuclease treated groups, and harvested 24 hours after electroporation of the nuclease treated groups.

[0353] For Western blot analysis, lysates were centrifuged and the supernatant was transferred to a new tube and placed on ice. Protein concentration was determined by BCA assay (Pierce) and 15 μg of total protein per sample was loaded into each well of the gel in sample buffer + DTT (NuPage) and incubated at 90°C for 10 minutes. 5 μg of each sample was loaded into each well of the gel. Individual mock samples from the 24 hour time point after electroporation were used as controls. After electrophoresis, samples were transferred (NuPage Electrophoresis System and Reagents) to PVDF membranes (Novex). Membranes were blocked with 5% nonfat dry milk in TBS-T and stained with primary antibody:

[0354] Blot Primary Antibody

[0355] A Rabbit polyclonal anti-nuclease (proprietary to Precision BioSciences, used at 1 :6500)

[0356] B Mouse anti-B-actin (Sigma, used at 1 :15000)

[0357] Membranes were washed 6 times and then incubated with the appropriate secondary antibody:

[0358] Blot Secondary Antibody

[0359] A Goat anti-rabbit HRP (Invitrogen, used at 1 :50000)

[0360] B Goat anti-mouse HRP (Invitrogen, used at 1 :75000)

[0361] After the wash steps, membranes were exposed to ECL Prime (Amersham), wrapped in Saran Wrap and images were captured using a UVP ChemiDoc-It 815 Imager.

[0362] As Figure 17As expected, no nuclease expression was detected in the simulated samples. In samples electroporated with mRNA encoding either TRC1-2x.87EE or TRC1-2L.1592 nucleases, the nuclease proteins were highly expressed at the earliest time point analyzed at 6 hours post-electroporation. The proteins remained detectable at 24 hours post-electroporation; however, expression of both nucleases was observed to be significantly lower at 6 hours post-electroporation, while at that time point TRC1-2L.1592 was significantly lower than TRC1-2x.87EE. In samples treated with TRC1-2L.1592 mRNA, the nuclease proteins were undetectable at 48 hours post-electroporation or subsequent time points, while TRC1-2x.87EE protein expression was still detectable at that time point. Actin expression was consistent across all samples and time points, indicating that an equal amount of protein was added to each sample.

[0363] These studies indicate that the TRC1-2x.87EE and TRC1-2L.1592 nucleases were expressed at high levels 6 hours after mRNA electroporation. However, the expression of TRC1-2L.1592 in T cells decreased more rapidly than that of TRC1-2x.87EE. Figure 11 As shown, TRC 1-2L.1592 did not exhibit reduced gene editing efficiency compared to TRC 1-2x.87EE, despite its shorter expression duration. The retention of high gene editing activity while reducing expression duration is an ideal characteristic of TRC 1-2L.1592 and represents an unexpected and favorable improvement compared to TRC 1-2x.87EE, as these characteristics are associated with TRC 1-2L.1592's enhanced (i.e., increased) tolerance and higher T cell proliferation capacity, as well as lower off-target activity, compared to TRC 1-2x.87EE.

[0364] Example 3

[0365] Evaluation of TRC 1-2 wide-ranging nucleases for CAR T production

[0366] Further evaluation of the TRC 1-2L.1592 macronuclease during large-scale process was conducted to determine whether the large-scale production of CAR T cells was improved compared to the first-generation TRC 1-2x.87EE macronuclease.

[0367] The large scale process for generating allogeneic CAR T cells with TRC 1-2x.87EE begins with a fresh Leukopak from a healthy pre-qualified donor. The Leukopak product is washed to remove platelets prior to immunomagnetic enrichment of target T cells. The enriched T cells are then washed into growth media and activated using an activation reagent. After a 3 day activation period, the cells are washed and concentrated in electroporation buffer. mRNA encoding TRC 1-2x.87EE is added and the mixture of cells and mRNA is processed through an electroporation device. The electroporated cells are diluted with growth media containing an AAV vector encoding a CAR insert gene. After an expansion period, the cells are collected on day 8 and subjected to immunomagnetic depletion of the CD3 positive population. After depletion, the target CD3 negative cells are expanded for an additional period of time in growth media. Finally, the cells are collected on day 13, washed, and concentrated into a cryoprotectant solution and frozen. The large scale process for generating allogeneic CAR T cells with TRC 1-2L.1592 is essentially the same as described for TRC 1-2x.87EE, except that the growth media formulation in the TRC 1-2L.1592 trial is animal origin free (AOF).

[0368] The total number of viable cells is determined at key time points during the production process. Figure 18 The cell number is comparable from day 0 to the day 8 depletion step. However, due to the significantly higher T cell receptor knockdown efficiency of TRC 1-2L.1592, the depletion step in the TRC 1-2L.1592 process advantageously recovers more than twice the number of cells recovered in the TRC 1-2x.87EE process. The expansion rate between day 8 and day 13 is similar, resulting in approximately twice as many total viable cells on day 13.

[0369] The CD3 knockdown efficiency (i.e., an indicator of endogenous T cell receptor knockdown) is determined by flow cytometry on day 8 of each production process. Figure 19 Unexpectedly, the percentage of CD3 negative, gene edited cells (in total viable cells) is nearly 20% higher in the TRC 1-2L.1592 process than in the TRC 1-2x.87EE process.

[0370] Finally, the CAR knockin efficiency is measured by flow cytometry at three key time points in each production process. Figure 20). Surprisingly, the percentage of CAR positive transduced cells (in CD3 negative cells) during the TRC 1-2L.1592 treatment process was about 25% higher compared to the TRC 1-2x.87EE treatment process. The percentage of CAR knock-in was stable between day 8 and the end of the process at day 13 for both processes, resulting in a similarly higher percentage of CAR positive cells at the end of the TRC 1-2L.1592 treatment process.

[0371] In summary, these studies surprisingly show that the TRC 1-2L.1592 nuclease significantly improves both the quantity and quality of the final allogeneic cell therapy product. TRC 1-2L.1592 more efficiently knocks out the endogenous T cell receptor, resulting in a larger population of gene edited CD3 negative cells, increasing the yield of the entire production process by about a factor of two. In addition, TRC 1-2L.1592 potentially provides an improved environment for homologous recombination at the targeted double strand break with the CAR gene insert, as evidenced by the improved CAR knock-in efficiency. The increase in the percentage of CAR positive cells results in a significantly higher drug product purity with fewer CAR negative cells.

Claims

1. An engineered wide-range nuclease, said wide-range nuclease binding to and cleaving the recognition sequence consisting of SEQ ID NO: 5 within the human T cell receptor (TCR) α constant region gene, wherein said engineered wide-range nuclease consists of the amino acid sequence of SEQ ID NO:

7.

2. A polynucleotide comprising a nucleic acid sequence encoding the engineered wide-range nuclease of claim 1.

3. The polynucleotide of claim 2, wherein the polynucleotide is mRNA.

4. A recombinant DNA construct comprising the polynucleotide according to claim 2.

5. A viral vector comprising the polynucleotide according to claim 2.

6. The viral vector according to claim 5, wherein the viral vector is a recombinant adeno-associated virus (AAV) vector.

7. The use of the engineered large-scale nuclease according to claim 1 or the polynucleotide according to claim 2 or 3 for producing genetically modified eukaryotic cells by disrupting target sequences in eukaryotic chromosomes. The engineered wide-range nuclease wherein the target sequence is disrupted by non-homologous end joining at the cleavage site in the chromosome at the recognition sequence consisting of SEQ ID NO:

5.

8. The use of the engineered large-scale nuclease according to claim 1 or the polynucleotide according to claim 2 or 3, and the nucleic acid comprising the target sequence for producing genetically modified eukaryotic cells, wherein the genetically modified eukaryotic cells comprise an exogenous target sequence inserted into the chromosome of the eukaryotic cells. The engineered wide-range nuclease generates a cleavage site in the chromosome at the recognition sequence consisting of SEQ ID NO: 5; and the target sequence is inserted into the chromosome at the cleavage site.

9. The use according to claim 8, wherein the nucleic acid containing the target sequence is introduced into the eukaryotic cell via a viral vector.

10. The use according to claim 9, wherein the viral vector is a recombinant AAV vector.

11. The use according to any one of claims 8-10, wherein the target sequence comprises a coding sequence for a chimeric antigen receptor or an exogenous T-cell receptor.

12. The use according to any one of claims 7-11, wherein the eukaryotic cell is a human T cell or a cell derived therefrom, or a human NK cell or a cell derived therefrom.

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