Optimized engineered nuclease with specificity for human T cell receptor alpha constant region gene

Abstract

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

Description

[0001] This application is a divisional application of the patent application with the application number 201980037689.6, the application date of April 11, 2019, and the title of "Optimized Engineered Nucleases Specific for the Human T-Cell Receptor Alpha Constant Region Gene". Technical Field

[0002] 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 the human T-cell receptor alpha constant region gene. The present invention also relates to the use of such recombinant meganucleases in methods for producing genetically modified T cells and to methods for treating diseases including cancer in a subject using such cells.

[0003] Reference to the Sequence Listing Submitted as a Text File via EFS-WEB

[0004] This application contains a sequence listing that has been submitted in ASCII format via EFS-Web, the entire content of which is incorporated herein by reference. The ASCII copy created on April 11, 2019, is named P109070028WO00-SEQ and is 2,700 bytes in size. Background of the Invention

[0006] T-cell adoptive immunotherapy is a promising cancer treatment approach. This strategy utilizes isolated human T cells that have been genetically modified to enhance their specificity for specific tumor-associated antigens. Genetic modification can involve the expression of chimeric antigen receptors or exogenous T-cell receptors to transplant antigen specificity onto the T cells. In contrast to exogenous T-cell receptors, chimeric antigen receptors obtain their specificity from the variable domains of monoclonal antibodies. Thus, T cells expressing chimeric antigen receptors (CAR T cells) induce tumor immunoreactivity in a major histocompatibility complex-unrestricted manner. T-cell adoptive immunotherapy has been used as a clinical therapy for many cancers, including B-cell malignancies (such as 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.

[0007] Although it has potential use as a cancer treatment, adoptive immunotherapy with CAR T cells is limited in part by the expression of endogenous T cell receptors on the cell surface. After administration to an allogeneic patient, CAR T cells expressing endogenous T cell receptors can recognize major and minor histocompatibility antigens, which may 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 re-infused into the same patient. The autologous approach provides immune 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.

[0008] Accordingly, it would be advantageous to develop "off-the-shelf" CAR T cells prepared using T cells from third-party, healthy donors, which have reduced expression of endogenous T cell receptors and do not trigger GVHD upon administration. Such products could be generated and validated prior to diagnosis and could be supplied to patients as soon as necessary. Thus, there is a need to develop allogeneic CAR T cells lacking endogenous T cell receptors to prevent the occurrence of GVHD.

[0009] Genetic modification of genomic DNA can be carried out using site-specific, rare-cutting endonucleases that have been engineered to recognize DNA sequences in target loci. Homing endonucleases are a group of naturally occurring nucleases that recognize cleavage sites of 15 - 40 base pairs that are 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 promote homologous recombination or gene insertion at specific locations in the host genome by generating double-strand breaks in the chromosome, which recruits the cellular DNA repair machinery (Stoddard (2006), Q. Rev. Biophys. 38:49 - 95). Homing endonucleases are generally divided 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 to be monomers.

[0010] I-CreI (SEQ ID NO:1) is a member of the LAGLIDADG (SEQ ID NO:2) family of homing endonucleases that 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 has been 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).

[0011] As first described in WO 2009 / 059195, I-CreI and its engineered derivatives are typically dimeric, but can be fused into a single polypeptide using a short peptide linker that joins 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, functional "single-chain" meganucleases can be expressed from a single transcript.

[0012] The use of nucleases to disrupt the expression of endogenous TCR has been disclosed, including the use of small hairpin RNAs, 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).

[0013] The specific use of engineered meganucleases to cleave DNA targets in the human TCRα constant region gene has also been previously disclosed. For example, International Publication No. WO 2014 / 191527 discloses variants of the I-OnuI meganuclease that have also been engineered to target a recognition sequence within exon 1 of the TCRα constant region gene (SEQ ID NO: 3 disclosed in '527). Although the '527 publication discusses that chimeric antigen receptors can be expressed in TCR knockout cells, the authors did not disclose inserting the CAR coding sequence into the meganuclease cleavage site.

[0014] In addition, in International Publication Nos. WO 2017 / 062439 and WO 2017 / 062451, the applicant discloses engineered meganucleases that are specific for recognition sequences in exon 1 of the TCRα constant region gene. These include the "TRC 1-2 meganuclease" that is specific for the TRC 1-2 recognition sequence (SEQ ID NO: 5) in exon 1. The '439 and '451 publications also disclose methods for targeted insertion of a CAR coding sequence or an exogenous TCR coding sequence into the TCR 1-2 meganuclease cleavage site.

[0015] In the present invention, the applicant has improved the nucleases and methods taught in the prior art. Through extensive experimentation, the applicant has generated novel second-generation TRC 1-2 meganucleases that contain unique, unpredictable residue combinations and 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 duration in cells after mRNA expression, are superior in vitro function when used to generate CAR T cells (e.g., enhanced / increased TCR knockout, enhanced / increased CAR knock-in, enhanced / increased CAR T expansion, improved CAR T cell phenotype, etc.), and produce an improved CAR T cell population when used in a comprehensive CAR T cell manufacturing process. SUMMARY OF THE INVENTION

[0016] The present invention provides engineered meganucleases that recognize and cleave a recognition sequence within the first exon of the human T cell receptor (TCR) α constant region gene (SEQ ID NO: 3). Such meganucleases can be used to disrupt the TCRα constant region gene and thus disrupt the expression and / or function of the cell surface TCR. Meganuclease cleavage can disrupt gene function through mutagenesis by non-homologous end joining or by facilitating the introduction of a foreign polynucleotide into the gene via homologous recombination. In some embodiments, the introduced foreign polynucleotide contains a nucleic acid sequence encoding a chimeric antigen receptor (CAR) such that the meganuclease can be used to generate allogeneic CAR T cells lacking 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 duration in cells (e.g., after mRNA expression) and / or enhanced (i.e., increased) modification efficiency of the TCRα constant region gene. In addition, cells that have been genetically modified with the presently disclosed engineered meganucleases exhibit improved characteristics compared to cells that have been genetically modified with the TRC1-2x.87EE meganuclease, including reduced off-target cleavage and its effects, reduced duration of the meganuclease in cells, enhanced (i.e., increased) CAR T expression, and lower differentiation. Further, cell populations into which the presently disclosed meganuclease (or nucleic acid encoding it) has been introduced have a greater percentage of modified cells and a greater percentage of less differentiated cells compared to those cell populations into which the TRC 1-2x.87EE meganuclease (or nucleic acid encoding it) has been introduced.

[0017] The present invention further provides methods, which include delivering an engineered meganuclease protein or a gene encoding an engineered meganuclease to a eukaryotic cell to generate a genetically modified eukaryotic cell. Accordingly, there are further provided genetically modified eukaryotic cells and populations thereof, as well as pharmaceutical compositions comprising the genetically modified eukaryotic cells and populations thereof. There are also provided methods of immunotherapy for treating cancer by administering a genetically modified T cell or a population thereof, wherein the T cell expresses a receptor for a tumor-specific antigen (e.g., a CAR or an exogenous TCR).

[0018] Thus, in one aspect, the present invention provides an engineered meganuclease that recognizes and cleaves the TRC 1-2 recognition sequence (SEQ ID NO: 5) in exon 1 of the human TCRα constant region gene (SEQ ID NO: 3). The engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to the first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and wherein the second subunit binds to the second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region that has 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 higher sequence identity with the amino acid sequence corresponding to residues 24-79 of the currently disclosed TRC 1-2L.1592 (the amino acid sequence of which is shown 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 higher sequence identity with the amino acid sequence corresponding to residues 24-79 of the currently disclosed TRC1-2L.1775 meganuclease (the amino acid sequence of which is shown in SEQ ID NO: 8).

[0019] In some embodiments, the HVR2 region comprises the amino acid sequence corresponding to residues 24-79 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.

[0020] In some embodiments, the HVR2 region comprises the residues corresponding to residues 24, 26, 42, 44, 46, 48, 50, 70, 71, 72 and 73 of SEQ ID NO: 7.

[0021] 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.

[0022] 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.

[0023] In some embodiments, the HVR2 region comprises residues corresponding to residues 48, 50, 71, 72 and 73 of SEQ ID NO: 7.

[0024] In some embodiments, the HVR2 region comprises residues corresponding to residues 48 and 50 of SEQ ID NO: 8.

[0025] 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.

[0026] In some embodiments, the HVR2 region comprises Y, R, K or D at the residue corresponding to residue 66 of SEQ ID NO: 7 or 8.

[0027] In some embodiments, the HVR2 region comprises residues 24 - 79 of SEQ ID NO: 7 or 8.

[0028] In certain 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 higher sequence identity with the 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 with the 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 with the amino acid sequence corresponding to residues 7 - 153 of SEQ ID NO: 8.

[0029] In some embodiments, the second subunit comprises the amino acid sequence corresponding to residues 7-153 of SEQ ID NO: 7 or 8, having 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.

[0030] In some embodiments, the second subunit comprises G, S, or A at the residue corresponding to residue 19 of SEQ ID NO: 7 or 8.

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

[0032] In some embodiments, the second subunit comprises the residue corresponding to residue 80 of SEQ ID NO: 7 or 8.

[0033] In some embodiments, the second subunit comprises the residue corresponding to residue 139 of SEQ ID NO: 7 or 8.

[0034] In certain embodiments, the second subunit comprises residues 7-153 of SEQ ID NO: 7 or 8.

[0035] 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 higher sequence identity to the amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7 or 8. In some embodiments, the HVR1 region comprises the amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7 or 8, having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.

[0036] In some embodiments, the HVR1 region comprises the residues corresponding to residues 219 and 231 of SEQ ID NO: 7.

[0037] In some embodiments, the HVR1 region comprises the 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.

[0038] In some embodiments, the HVR1 region contains Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 7 or 8.

[0039] In certain embodiments, the HVR1 region contains residues 215-270 of SEQ ID NO: 7 or 8.

[0040] In some embodiments, the first subunit contains 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 higher 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 contains 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 certain embodiments, the first subunit contains the amino acid sequence corresponding to residues 198-344 of SEQ ID NO: 7 or 8, with 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.

[0041] In some embodiments, the first subunit contains G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 7 or 8.

[0042] In some embodiments, the first subunit contains E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 7 or 8.

[0043] In some embodiments, the first subunit contains the residue corresponding to residue 271 of SEQ ID NO: 7 or 8.

[0044] In certain embodiments, the first subunit contains residues 198-344 of SEQ ID NO: 7 or 8.

[0045] 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 higher sequence identity with the amino acid sequence corresponding to residues 198 - 344 of SEQ ID NO: 7 or 8, while 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 higher sequence identity with the amino acid sequence corresponding to residues 7 - 153 of SEQ ID NO: 7 or 8. In certain embodiments, the first subunit of the engineered meganuclease has at least 99% sequence identity with the amino acid sequence corresponding to residues 198 - 344 of SEQ ID NO: 7 or 8, while the second subunit comprises an amino acid sequence having at least 93% sequence identity with 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 may each contain 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 of SEQ ID NO: 7 and residues 7 - 153 of SEQ ID NO: 8, respectively.

[0046] In some embodiments, the engineered meganuclease comprises a linker, wherein the linker covalently joins the first subunit and the second subunit.

[0047] In some embodiments, the engineered meganuclease has an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 7 or 8. In some embodiments, the engineered meganuclease has an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 7. In some embodiments, the engineered meganuclease has an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 8.

[0048] In certain embodiments, the engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 7 or 8.

[0049] In some embodiments, compared to the TRC 1-2x.87EE meganuclease as shown in SEQ ID NO: 9, the engineered meganuclease exhibits at least one of the following optimized characteristics: improved (i.e., increased) specificity, reduced intracellular duration, and enhanced (i.e., increased) modification efficiency of the human TCRα constant region gene.

[0050] In certain embodiments, the engineered meganuclease that recognizes and cleaves the recognition sequence comprising SEQ ID NO: 5 within the human TCRα 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 higher sequence identity with 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 higher sequence identity with 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 higher sequence identity with 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 higher sequence identity with the amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7 or 8.

[0051] In certain embodiments, an engineered meganuclease that recognizes and cleaves a recognition sequence comprising SEQ ID NO: 5 within the human TCRα 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 higher 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 higher 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; 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 higher 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 higher 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, 68, 70, 75 and 77 of SEQ ID NO: 7 or 8. In these embodiments, the HVR2 region may 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.

[0052] In certain embodiments, an engineered meganuclease that recognizes and cleaves the recognition sequence comprising SEQ ID NO:5 within the human TCRα 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 higher 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 higher 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 higher 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 higher 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.

[0053] In other embodiments, an engineered meganuclease that recognizes and cleaves the recognition sequence comprising SEQ ID NO:5 within the human TCRα 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 higher sequence identity to residues 198-344 of SEQ ID NO:7 or 8; (b) an 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 higher sequence identity to residues 7-153 of SEQ ID NO:7 or 8; and (b) an HVR2 region having the amino acid sequence corresponding to residues 24-79 of SEQ ID NO:7 or 8.

[0054] In another aspect, the invention provides a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease as described herein.

[0055] In some embodiments, the polynucleotide is mRNA.

[0056] In other embodiments, the mRNA is a polycistronic mRNA encoding an engineered meganuclease and at least one additional polypeptide or nucleic acid described herein.

[0057] In another aspect, the invention provides a recombinant DNA construct comprising a polynucleotide described herein.

[0058] 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 virus (AAV) vector. In specific embodiments, the viral vector is a recombinant AAV vector.

[0059] In another aspect, the invention provides a viral vector comprising a polynucleotide described herein.

[0060] 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.

[0061] In another aspect, the invention provides a method of generating a genetically modified eukaryotic cell comprising an exogenous target sequence inserted into the chromosome of the eukaryotic cell. The method includes introducing one or more nucleic acids into the eukaryotic cell, the 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 target sequence; wherein the engineered meganuclease creates a cleavage site in the chromosome at the recognition sequence comprising SEQ ID NO:5; and wherein the target sequence is inserted into the chromosome at the cleavage site.

[0062] In certain embodiments of the method, the second nucleic acid further comprises a sequence homologous to the sequences flanking the cleavage site, and the target sequence is inserted at the cleavage site by homologous recombination.

[0063] In certain embodiments of the method, the second nucleic acid does not comprise a sequence homologous to the sequences flanking the cleavage site, and the target sequence is inserted at the cleavage site by non-homologous insertion.

[0064] In certain embodiments of the method, the cell surface expression of an endogenous T cell receptor (e.g., an α / β T cell receptor) is reduced compared to an unmodified control cell.

[0065] 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.

[0066] In some embodiments of the method, the target sequence comprises a coding sequence of a chimeric antigen receptor or an exogenous T cell receptor. In certain 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.

[0067] In some embodiments of the method, at least the first nucleic acid is introduced into the eukaryotic cell by mRNA.

[0068] In certain embodiments of the method, at least the second nucleic acid is introduced into the eukaryotic cell by a viral vector. In specific embodiments of the method, the viral vector is an adenovirus vector, a lentivirus vector, a retrovirus vector, or an AAV vector. In a particular embodiment of the method, the viral vector is a recombinant AAV vector.

[0069] In another aspect, the present invention provides a method for generating a genetically modified eukaryotic cell, which comprises inserting an exogenous target sequence into the chromosome of the eukaryotic cell. The method includes: (a) introducing the engineered meganuclease described herein into the eukaryotic cell; and (b) introducing a nucleic acid comprising the target sequence into the eukaryotic cell; wherein the engineered meganuclease generates a cleavage site at the recognition sequence of SEQ ID NO: 5 in the chromosome; and wherein the target sequence is inserted into the chromosome at the cleavage site.

[0070] In certain embodiments of the method, the nucleic acid further comprises a sequence homologous to the sequence flanking the cleavage site, and the target sequence is inserted at the cleavage site by homologous recombination.

[0071] In certain embodiments of the method, the nucleic acid does not comprise a sequence homologous to the sequence flanking the cleavage site, and the target sequence is inserted at the cleavage site by non-homologous insertion.

[0072] In certain embodiments of the method, compared with unmodified control cells, the cell surface expression of the endogenous T cell receptor (such as the α / β T cell receptor) is reduced.

[0073] 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.

[0074] In some embodiments of the method, the target sequence comprises a coding sequence of a chimeric antigen receptor or an exogenous T cell receptor. In certain 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.

[0075] In certain embodiments of the method, nucleic acids are introduced into eukaryotic cells via a viral vector. In specific embodiments of the method, the viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an AAV vector. In particular embodiments of the method, the viral vector is a recombinant AAV vector.

[0076] In another aspect, the present invention provides a method for generating genetically modified eukaryotic cells by disrupting a target sequence in a eukaryotic cell chromosome. The method comprises introducing a nucleic acid encoding an engineered meganuclease as described herein into a eukaryotic cell, wherein the engineered meganuclease is expressed in the eukaryotic cell, and wherein the engineered meganuclease generates a cleavage site in the chromosome at the recognition sequence comprising SEQ ID NO: 5, and wherein the target sequence is disrupted by non-homologous end joining at the cleavage site.

[0077] In certain embodiments of the method, the cell surface expression of an endogenous T cell receptor (e.g., an α / β T cell receptor) is reduced compared to unmodified control cells.

[0078] 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.

[0079] In some embodiments of the method, the nucleic acid is introduced into the eukaryotic cell via mRNA.

[0080] In another aspect, the present invention provides a method for generating genetically modified eukaryotic cells by disrupting a target sequence in a eukaryotic cell chromosome. The method comprises introducing an engineered meganuclease as described herein into a eukaryotic cell, wherein the engineered meganuclease generates a cleavage site in the chromosome at the recognition sequence comprising SEQ ID NO: 5, and wherein the target sequence is disrupted by non-homologous end joining at the cleavage site.

[0081] In certain embodiments of the method, the cell surface expression of an endogenous T cell receptor (e.g., an α / β T cell receptor) is reduced compared to unmodified control cells.

[0082] 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.

[0083] In another aspect, the present invention provides a genetically modified eukaryotic cell comprising a modified human T cell receptor alpha constant region gene in its genome, wherein the modified human T cell receptor alpha constant region gene comprises an exogenous target sequence within exon 1 of SEQ ID NO: 5 inserted into the T cell receptor alpha constant region, and wherein the genetically modified eukaryotic cell is prepared by the method described herein using the engineered meganuclease described herein.

[0084] 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.

[0085] In certain embodiments, the target sequence comprises the coding sequence of a chimeric antigen receptor or an exogenous T cell receptor. In a particular embodiment, the chimeric antigen receptor or exogenous T cell receptor comprises an extracellular ligand-binding domain specific for a tumor-specific antigen.

[0086] In a particular embodiment, the cell surface expression of the endogenous T cell receptor (e.g., alpha / beta T cell receptor) is reduced on the genetically modified eukaryotic cell as compared to an unmodified control cell.

[0087] In a particular embodiment, the genetically modified eukaryotic cell comprises a reduced off-target effect of the engineered meganuclease, and / or a reduced duration of the engineered meganuclease in the cell, as compared to the TRC 1-2x.87EE meganuclease as shown in SEQ ID NO: 9.

[0088] In another aspect, the present invention provides a genetically modified eukaryotic cell comprising a chromosome having a target sequence disrupted at the 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 prepared by the method described herein using the engineered meganuclease described herein.

[0089] 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.

[0090] In a particular embodiment, the cell surface expression of the 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.

[0091] In certain embodiments, compared to the TRC 1-2x.87EE meganuclease as shown in SEQ ID NO:9, the genetically modified eukaryotic cells comprise a reduced off-target effect of the engineered meganuclease and / or a reduced duration in the cells.

[0092] In another aspect, the invention provides a population of genetically modified eukaryotic cells, which comprises a plurality of the genetically modified eukaryotic cells described herein.

[0093] 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 the genetically modified eukaryotic cells described herein.

[0094] In certain 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.

[0095] In certain embodiments, the genetically modified eukaryotic cells of the population comprise a chimeric antigen receptor on the cell surface 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.

[0096] In certain embodiments, compared to unmodified control cells, the genetically modified eukaryotic cells of the population have a reduced cell surface expression of endogenous T cell receptors (e.g., α / β T cell receptors).

[0097] In another aspect, the invention provides a pharmaceutical composition for use in treating a disease in a subject in need thereof, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a therapeutically effective amount of the genetically modified eukaryotic cells or a population thereof described herein.

[0098] In certain embodiments, the genetically modified eukaryotic cells are genetically modified human T cells or cells derived therefrom, or genetically modified NK cells or cells 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.

[0099] In some embodiments, the exogenous target sequence present in a genetically modified T cell or a population thereof comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor. In certain specific embodiments, the chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand-binding domain that is specific for a tumor-specific antigen.

[0100] In some embodiments, the cell surface expression of the endogenous T cell receptor (e.g., α / β T cell receptor) on the genetically modified eukaryotic cell is reduced compared to an unmodified control cell.

[0101] In another aspect, the present invention provides a lipid nanoparticle or a 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.

[0102] In another aspect, the present invention 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 a population thereof as described herein.

[0103] In some embodiments, the method comprises administering to the subject a pharmaceutical composition as described herein.

[0104] In certain embodiments, the method is an immunotherapy for treating 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 target sequence present in the genetically modified eukaryotic cell comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor, the receptor comprising an extracellular ligand-binding domain that is specific for a tumor-specific antigen, and the cell surface expression of the endogenous T cell receptor (e.g., α- / β T cell receptor) is reduced on the genetically modified eukaryotic cell compared to an unmodified control cell.

[0105] In some embodiments of the method, the cancer is selected from carcinoma, lymphoma, sarcoma, blastoma, and leukemia.

[0106] In certain embodiments of the method, the cancer is selected from cancers 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 lymphoma.

[0107] In a specific embodiment of the method, the cancers of B cell origin are selected from B-lineage acute lymphoblastic leukemia, B cell chronic lymphocytic leukemia, B cell non-Hodgkin lymphoma, and multiple myeloma.

[0108] In certain embodiments of the method, the subject can be a mammal, such as a human.

[0109] In another aspect, the invention provides a genetically modified cell or population thereof as described herein for use as a medicament. The invention also provides the use of a genetically modified cell or population thereof as described herein in the preparation of a medicament for treating a disease in a subject in need thereof. In one such aspect, the medicament can be used to treat cancer.

[0110] In another aspect, the invention provides a genetically modified cell or population thereof as described herein for treating a disease, preferably for treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 . The TRC 1-2 recognition sequence in the human T cell receptor alpha constant region gene. The TRC 1-2 recognition sequence targeted by the engineered meganuclease of the invention comprises two recognition half-sites. Each recognition half-site comprises 9 base pairs, separated by a central sequence of 4 base pairs. The TRC 1-2 recognition sequence (SEQ ID NO: 5) comprises two recognition half-sites designated TRC1 and TRC2.

[0112] Figure 2 . The engineered meganuclease of the invention comprises two subunits, wherein the first subunit comprising the HVR1 region binds to the first recognition half-site (e.g., TRC1), while the second subunit comprising the HVR2 region binds to the second recognition half-site (e.g., TRC2). In embodiments in which the engineered meganuclease is a single-stranded meganuclease, the first subunit comprising the HVR1 region can be positioned as the N-terminal or C-terminal subunit. Similarly, the second subunit comprising the HVR2 region can be positioned as the N-terminal or C-terminal subunit.

[0113] Figure 3. Schematic of the reporter assay for evaluating the engineered meganucleases of the present invention in CHO cells. A CHO cell line was generated in which the reporter gene cassette was stably integrated into the cell genome. The reporter gene cassette contains, in 5' to 3' order: the SV40 early promoter; the 2 / 3 of the 5' side of the GFP gene; the recognition sequence of the engineered meganuclease of the present invention (e.g., the TRC 1-2 recognition sequence); the recognition sequence of the CHO 23 / 24 meganuclease (WO / 2012 / 167192); and the 2 / 3 of the 3' side of the GFP gene. In the absence of a DNA break inducer, cells stably transfected with this cassette do not express GFP. Meganucleases are introduced by transduction of plasmid DNA or mRNA encoding each meganuclease. When DNA breaks are induced at either meganuclease recognition sequence, the repeat regions of the GFP gene recombine with each other to generate 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.

[0114] Figure 4 . Efficiency of engineered meganucleases recognizing and cleaving the TRC 1-2 recognition sequence in the CHO cell reporter assay. The TRC 1-2L.1592, TRC 1-2L.1775 and TRC 1-2L.1843 meganucleases were engineered to target the TRC 1-2 recognition sequence (SEQ ID NO: 5) and were screened for efficacy in the CHO cell reporter assay. The results shown provide the percentage of cells observed to express GFP, which indicates the efficacy of each meganuclease in cleaving the target recognition sequence or the CHO 23 / 24 recognition sequence. A negative control (bs) and the first generation TRC 1-2x.87EE were further included in the assay for comparison. A) CHO reporter assay evaluating TRC 1-2L.1592. B) CHO reporter assay evaluating TRC 1-2L.1775. C) CHO reporter assay evaluating TRC 1-2L.1843.

[0115] Figure 5. In the CHO cell reporter gene assay, the efficiency of engineered meganucleases to recognize and cleave the TRC Off1 recognition sequence (SEQ ID NO: 16) and the TRC Off2 recognition sequence (SEQ ID NO: 17) was determined. mRNA encoding the TRC 1-2 meganuclease of the present invention was transfected into CHO reporter gene cells that contained either the counter-selected Off1 recognition sequence or the Off2 recognition sequence between direct repeats of GFP, as well as the CHO 23-24 recognition sequence. In each experiment, the second-generation meganuclease was compared to the first-generation TRC 1-2x.87EE meganuclease. A) Cleavage of off-target recognition sequences by TRC 1-2L.1592 and TRC 1-2x.87EE. B) Cleavage of off-target recognition sequences by TRC 1-2L.1775 and TRC 1-2x.87EE. C) Cleavage of off-target recognition sequences by TRC 1-2L.1843 and TRC 1-2x.87EE.

[0116] Figure 6 . In the CHO cell reporter gene assay, the efficiency of engineered meganucleases to recognize and cleave the TRC 1-2 recognition sequence was determined. The TRC 1-2x.87EE (first-generation), TRC 1-2L.1108 (intermediate), and TRC 1-2L.1469 (intermediate) meganucleases were engineered to target the TRC 1-2 recognition sequence (SEQ ID NO:5), and their efficacy was screened at 2, 5, and 7 days post-nucleofection in the CHO cell reporter gene assay to determine toxicity. The results shown provide the percentage of cells expressing GFP observed over a 7-day analysis period, which indicates the efficacy as a function of time of each meganuclease to cleave the target recognition sequence or the CHO 23 / 24 recognition sequence.

[0117] Figure 7 . In the CHO cell reporter gene assay, the efficiency of engineered meganucleases to recognize and cleave the TRC 1-2 recognition sequence was determined. The second-generation TRC1-2L.1592, TRC1-2L.1775, and TRC1-2L.1843 meganucleases were optimized to target the TRC1-2 recognition sequence (SEQ ID NO: 5), and their efficacy was screened at 2, 5, and 7 days post-nucleofection in the CHO cell reporter gene assay to determine toxicity. The analysis also included the first-generation TRC 1-2x.87EE meganuclease and the intermediate TRC1-2L.1469 meganuclease for comparison. The results shown provide the percentage of cells expressing GFP observed over the 7-day analysis, indicating the efficacy as a function of time of each meganuclease to cleave the target recognition sequence or the CHO 23 / 24 recognition sequence.

[0118] Figure 8 . In a CHO cell reporter gene assay, the efficiency of engineered meganucleases to recognize and cleave the TRC Off1 and Off2 recognition sequences. In a CHO cell reporter gene assay, the first-generation TRC 1-2x.87EE meganuclease, the intermediate TRC 1-2L.1469 meganuclease, and the second-generation TRC 1-2L.1592, TRC 1-2L.1775, and TRC 1-2L.1843 meganucleases were screened for efficacy at 2, 5, and 7 days post-nucleofection in CHO GFFP reporter cells containing the TRC Off1 (SEQ ID NO: 16) or Off2 (SEQ ID NO: 17) recognition sequences to determine toxicity. The results shown provide the percentage of GFP-expressing cells observed over a 7-day analysis period. A) Cleavage of the Off1 recognition sequence. B) Cleavage of the Off2 recognition sequence.

[0119] Figure 9 . Graphical visualization of oligonucleotide capture data as a measure of the number of potential off-target sites. For each off-target cleavage generated by a specific nuclease, the number of unique sequence reads of the probe oligonucleotide captured at that site was plotted. For each tested meganuclease (circled), the expected site (i.e., the TRC 1-2 recognition sequence) had the highest read count.

[0120] Figure 10 . Graphical visualization of oligonucleotide capture data, where off-target sites were plotted according to their aligned read count on the x-axis and the number of mismatched base pairs compared to the expected site was represented by color, with darker colors indicating a closer overall match between the off-target and expected binding sites. The boxes represent the regions of highest confidence.

[0121] Figure 11 . Table summarizing the in vitro analysis of CAR T cells generated using the first-generation TRC 1-2x.87EE meganuclease, the intermediate TRC 1-2L.1469 meganuclease, or the second-generation TRC 1-2L.1592, TRC 1-2L.1775, and TRC 1-2L.1843 meganucleases. The gene editing efficiency, post-editing expansion, and differentiation potential of the meganucleases were screened. CAR T cells were prepared from cells obtained from three different healthy human donors and the experiments were performed by three different operators.

[0122] Figure 12 . Graphical visualization of oligonucleotide capture data generated from T cell populations obtained from three different healthy human donors.

[0123] Figure 13. In 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 cell count at days 0, 4, and 8 post-editing. B) Total number of edited cells (i.e., TCR-negative) at days 0, 4, and 8 post-editing. C) Total number of TCR-negative / CAR-positive cells at days 0, 4, and 8 post-editing.

[0124] Figure 14 . Expansion of CAR T cells after co-culture with antigen-bearing target cells. CAR T cells were co-cultured with the CD19+ tumor lines Raji or Nalm6 at E:T ratios of 1:1 and 1:2 for 5 days and their expansion was evaluated. The cell input numbers are indicated by the dashed lines.

[0125] Figure 15 . Expansion of CAR T cells after co-culture with antigen-bearing target cells. CAR T cells were co-cultured with the Raji CD19+ tumor cell line at an E:T ratio of 1:2 for 5 days and their expansion was evaluated. A) Total number of CAR-positive cells in the culture after co-culture with Raji cells. B) Total number of CD19-positive cells remaining in the culture after co-culture of CAR T cells with Raji cells.

[0126] Figure 16 . Secretion of CAR T cell cytokines into the culture supernatant after 2 days of co-culture with antigen-bearing target cells. CAR T cells were co-cultured with the CD19+ tumor cell lines Raji or Nalm6 at E:T ratios of 1:1 and 1:2 and cytokine secretion was evaluated. CD19-negative K562 myeloid leukemia cells were used as controls. A) IL-2 secretion. B) TNF-α secretion. C) INF-γ secretion. D) Secretion of granzyme B. E) Perforin secretion.

[0127] 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 meganuclease and then transduced with a recombinant AAV6 vector carrying a donor template encoding an anti-CD19 CAR designed to insert at the TRC 1-2 locus. 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 medium as the nuclease-treated groups and harvested 24 hours after electroporation of the nuclease-treated groups.

[0128] Figure 18. Total number of viable cells at days 0, 3, 8 (before and after CD3-positive cell depletion), and 13 during large-scale CAR T manufacturing runs using TRC 1-2x.87EE or TRC 1-2L.1592.

[0129] Figure 19 . Total number of viable CD3-negative cells on day 8 during large-scale CAR T manufacturing runs using TRC 1-2x.87EE or TRC 1-2L.1592.

[0130] Figure 20 . Percentage of CAR-positive CD3-negative cells on day 8 (before and after CD3-positive cell depletion) and day 13 during large-scale CAR T manufacturing runs using TRC 1-2x.87EE or TRC 1-2L.1592.

[0131] Sequence Description

[0132] SEQ ID NO: 1 lists the amino acid sequence of the wild-type I-CreI meganuclease from Chlamydomonas reinhardtii.

[0133] SEQ ID NO: 2 lists the amino acid sequence of the LAGLIDADG motif.

[0134] SEQ ID NO: 3 lists the nucleic acid sequence of the human T cell receptor alpha constant region gene (NCBI Gene ID number 28755).

[0135] SEQ ID NO: 4 lists the amino acid sequence of the polypeptide encoded by the human T cell receptor alpha constant region gene.

[0136] SEQ ID NO: 5 lists the nucleic acid sequence of the sense strand of the TRC 1-2 recognition sequence.

[0137] SEQ ID NO: 6 lists the nucleic acid sequence of the antisense strand of the TRC 1-2 recognition sequence.

[0138] SEQ ID NO: 7 lists the amino acid sequence of the TRC 1-2L.1592 meganuclease.

[0139] SEQ ID NO: 8 lists the amino acid sequence of the TRC 1-2L.1775 meganuclease.

[0140] SEQ ID NO: 9 lists the amino acid sequence of the TRC 1-2x.87EE meganuclease.

[0141] SEQ ID NO: 10 lists the amino acid sequence of the TRC1 binding subunit of the TRC 1-2L.1592 meganuclease.

[0142] SEQ ID NO: 11 lists the amino acid sequence of the TRC1 binding subunit of the TRC 1-2L.1775 meganuclease.

[0143] SEQ ID NO: 12 lists the amino acid sequence of the TRC1 binding subunit of the TRC 1-2x.87EE meganuclease.

[0144] SEQ ID NO: 13 lists the amino acid sequence of the TRC2 binding subunit of the TRC 1-2L.1592 meganuclease.

[0145] SEQ ID NO: 14 lists the amino acid sequence of the TRC2 binding subunit of the TRC 1-2L.1775 meganuclease.

[0146] SEQ ID NO: 15 lists the amino acid sequence of the TRC2 binding subunit of the TRC 1-2x.87EE meganuclease.

[0147] SEQ ID NO: 16 lists the nucleic acid sequence of the Off1 recognition sequence.

[0148] SEQ ID NO: 17 lists the nucleic acid sequence of the Off2 recognition sequence.

[0149] SEQ ID NO: 18 lists the amino acid sequence of the polypeptide linker. Detailed embodiments

[0150] 1.1 References and definitions

[0151] The patents and scientific literature referred to herein establish the knowledge available to those skilled in the art. The issued U.S. and non-U.S. patents, licensed applications, published U.S., non-U.S. and PCT applications, co-owned and co-pending unpublished U.S. patent applications, published foreign applications, and scientific, technical and medical references cited herein, including GenBank database sequences, public gene and protein database accession numbers or codes (and the nucleic acid and / or amino acid sequences associated therewith), are incorporated herein by reference to the extent as if each were specifically and individually indicated to be incorporated by reference.

[0152] The present invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. For example, features elucidated with respect to one embodiment may be incorporated into other embodiments, and features elucidated with respect to a particular embodiment may be deleted from that embodiment. Additionally, various changes and additions to the embodiments presented herein will be apparent to those skilled in the art in accordance with this disclosure without departing from the invention.

[0153] 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 invention belongs as of the priority date. The terms used in the description of the present invention herein are for the purpose of describing particular embodiments only and are not intended to limit the invention.

[0154] The entire contents of all publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference.

[0155] As used herein, "a / an" or "the" may mean one or more than one. For example, "a" cell may mean a single cell or multiple cells.

[0156] As used herein, the word "or" is used in the inclusive sense of "and / or" rather than the exclusive sense of "either / or" unless otherwise expressly indicated.

[0157] As used herein, the term "endonuclease" refers to an enzyme that cleaves phosphodiester bonds within a polynucleotide chain.

[0158] As used herein, with respect to double-stranded DNA, the terms "cut" or "cleave" refer to the hydrolysis of a phosphodiester bond within the backbone of a recognition sequence within a target sequence by an endonuclease, which results in a double-strand break within the target sequence, referred to herein as a "cut site". Depending on the endonuclease, the cut may produce a double-stranded fragment with blunt ends or a fragment with 5' or 3' base overhangs.

[0159] As used herein, the term "meganuclease" refers to an endonuclease that binds double-stranded DNA at an recognition sequence of greater than 12 base pairs. In some embodiments, the recognition sequence of the meganucleases of the present disclosure is 22 base pairs. The meganuclease can be an endonuclease derived from I-CreI and can refer to an engineered variant of I-CreI that has been modified, for example, in terms of DNA binding specificity, DNA cleavage activity, DNA binding affinity, or dimerization properties relative to native I-CreI. Methods for generating such modified variants of I-CreI are known in the art (e.g., WO 2007 / 047859, the entire content of which is 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". When expressed in the target cells described herein, particularly in human T cells, the meganucleases of the present disclosure are substantially non-toxic such that the cells can be transfected and maintained at 37°C without observing a significant detrimental effect on overall cell viability or a significant decrease in meganuclease cleavage activity when measured using the methods described herein.

[0160] As used herein, the term "single-chain meganuclease" refers to a polypeptide comprising a pair of nuclease subunits joined by a linker such that the subunits interact functionally as 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, single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. The single-chain meganuclease can be referred to as a "single-chain heterodimer" or "single-chain heterodimeric meganuclease", although it is not actually dimeric. For clarity, unless otherwise specified, the term "meganuclease" can refer to dimeric or single-chain meganucleases.

[0161] As used herein, the term "linker" refers to an exogenous peptide sequence that is used to link two large nuclease subunits into a single polypeptide. The linker can have a sequence found in a native protein, or can be an artificial sequence not found in any native 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 linker covered 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 higher sequence identity with 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.

[0162] As used herein, with respect to a protein, the terms "recombinant" or "engineered" refer to a protein having an altered amino acid sequence due to the application of genetic engineering techniques to the nucleic acid encoding the protein and to the cell or organism expressing the protein. With respect to a nucleic acid, the terms "recombinant" or "engineered" refer to a nucleic acid having 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. According to this definition, 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 to be recombinant.

[0163] 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. Wild-type alleles (i.e., polynucleotides) and polypeptides are distinguishable from mutant or variant alleles and polypeptides, which contain one or more mutations and / or substitutions relative to the wild-type sequence. Although 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. Wild-type nucleases are distinguishable from recombinant or non-naturally occurring nucleases. 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 used for comparison purposes.

[0164] As used herein, the term "genetically modified" refers to a cell or organism in which or in an ancestor of which the genomic DNA sequence has been intentionally modified by recombinant techniques. As used herein, the term "genetically modified" encompasses the term "transgenic".

[0165] As used herein with respect to recombinant proteins, the term "modification" refers to any insertion, deletion, or substitution of an amino acid residue in the recombinant sequence relative to a reference sequence (e.g., a wild-type or native sequence).

[0166] 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 a meganuclease, the recognition sequence contains a pair of inverted 9-base pair "half-sites" separated by four base pairs. In the case of a single-stranded meganuclease, 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 the meganuclease generates a 3' "overhang" of four base pairs. An "overhang" or "sticky end" is a short single-stranded DNA fragment that can be generated by cleavage of a double-stranded DNA sequence by an endonuclease. In the case of meganucleases derived from I-CreI and single-stranded meganucleases, the overhang contains bases 10-13 of the 22-base pair recognition sequence.

[0167] As used herein, the term "target site" or "target sequence" refers to a region of the chromosomal DNA of a cell that contains the recognition sequence of a nuclease.

[0168] 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 an "altered" binding affinity relative to a reference nuclease if the Kd of the nuclease for a reference recognition sequence changes by a statistically significant percentage or a biologically significant amount (e.g., at least 2x or 2x to 10x) increase or decrease.

[0169] 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 specific base pair sequence called the recognition sequence or only at a specific group of recognition sequences. This group of recognition sequences has 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.

[0170] As used herein, a nuclease has "altered" specificity if it binds and cleaves an recognition sequence that does not bind and cleave under physiological conditions by a reference nuclease (e.g., wild-type), or if the cleavage rate 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.

[0171] In some embodiments, compared to the TRC 1-2x.87EE meganuclease (the amino acid sequence of which is shown in SEQ ID NO: 9), 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). Thus, in certain embodiments, compared to the TRC 1-2x.87EE meganuclease, the engineered meganucleases disclosed herein exhibit reduced off-target cleavage. Any method known in the art can be used to measure off-target cleavage of meganucleases, 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, enrichment of direct in situ break labeling on streptavidin and next generation sequencing (BLESS), genome-wide unbiased DSB identification by sequencing (GUIDE-seq), and linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) described herein (see, e.g., Zischewski et al. (2017) Biotechnology Advances 35(1):95-104, the entire content of which is incorporated herein by reference in its entirety).

[0172] As used herein, the term "homologous recombination" or "HR" refers to the natural cellular process in which double-strand DNA breaks are 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 or an exogenous nucleic acid delivered to the cell.

[0173] As used herein, the term "non-homologous end joining" or "NHEJ" refers to a natural cellular process in which double-strand DNA breaks are repaired by the direct ligation 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 repair site. In some cases, cleavage at a target recognition sequence results in NHEJ at the target recognition site. Cleavage of a target site in a nuclease-induced gene coding sequence and subsequent DNA repair by NHEJ can introduce mutations (e.g., frameshift mutations) that disrupt gene function into the coding sequence. Thus, engineered nucleases can be used to effectively knock out genes in a cell population. As used herein, "disrupting a target sequence" refers to introducing a mutation (e.g., a frameshift mutation) that interferes with gene function and prevents the expression and / or function of the polypeptide / expression product encoded thereby.

[0174] As used herein, "homology arms" or "sequences homologous to the sequences flanking the meganuclease cleavage site" refer to sequences flanking the 5' and 3' ends of a nucleic acid molecule that facilitate insertion of the nucleic acid molecule into the cleavage site generated by a meganuclease. Generally, the length of the homology arms can be at least 50 base pairs, 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 higher sequence homology with their corresponding sequences in the genome.

[0175] As used herein, "chimeric antigen receptor" or "CAR" refers to an engineered receptor that confers or transfers specificity for an antigen to 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 that comprises one or more signaling domains and / or co-stimulatory domains.

[0176] 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 that provides specificity for a particular epitope or antigen (e.g., an epitope or antigen that is 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 desired antigen or epitope. In some embodiments, the scFv is murine, humanized, or fully human.

[0177] The extracellular domain of the chimeric antigen receptor may also contain an autoantigen (see Payne et al. (2016), Science 353(6295):179-184), which can be recognized by the autoantigen-specific B cell receptor on B lymphocytes, thereby guiding T cells to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases. Such CARs can be referred to as chimeric autoantibody receptors (CAARs), and their use is encompassed by the present invention.

[0178] The extracellular domain of the chimeric antigen receptor may also contain a naturally occurring ligand of the antigen of interest, or a fragment of a naturally occurring ligand that retains the ability to bind the antigen of interest.

[0179] The intracellular stimulatory domain may contain one or more cytoplasmic signaling domains that transmit an activation signal to the immune effector cell upon antigen binding. Such cytoplasmic signaling domains may include but are not limited to The intracellular stimulatory domain may also contain one or more intracellular co-stimulatory domains that transmit proliferation and / or cell survival signals upon ligand binding. Such intracellular co-stimulatory domains can be any domain known in the art and may 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 ligands that specifically bind to CD83, N1, N6, or any combination thereof.

[0180] The chimeric antigen receptor may 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 may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane polypeptide may be a subunit of the T cell receptor (i.e., the α, β, γ, or ζ polypeptide that constitutes the CD3 complex), the IL2 receptor p55 (α chain), p75 (β chain), or γ chain, the Fc receptor (e.g., Fcγ receptor III), or a subunit chain of a CD protein (e.g., the CD8α chain). Optionally, the transmembrane domain may be synthetic and may primarily comprise hydrophobic residues (e.g., leucine and valine).

[0181] A hinge region refers to any oligopeptide or polypeptide having the function of connecting a transmembrane domain to an extracellular ligand-binding domain. For example, the hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4 or CD28, or all or part of an antibody constant region. Optionally, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence. In certain instances, the hinge domain may comprise a portion of the human CD8α chain, the FcγRIIIa receptor, or IgG1.

[0182] As used herein, "exogenous T cell receptor" or "exogenous TCR" refers to a TCR whose sequence has been introduced into the genome of an immune effector cell (e.g., a human T cell) that may or may 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 cancer cells or other disease-causing cells or particles). Such exogenous T cell receptors may comprise an α and a β chain, or alternatively, may comprise a γ chain and a δ chain. The exogenous TCRs useful in the present invention can be specific for any antigen or epitope of interest.

[0183] As used herein, the term "reduced expression" refers to any reduction in the expression of an endogenous T cell receptor (e.g., an α / β T cell receptor) on the cell surface of a genetically modified T cell as compared to a control cell. The term reduced may also refer to a reduction in the percentage of cells in a cell population that express an endogenous polypeptide (i.e., an endogenous T cell receptor) on the cell surface as compared to a control cell population. Such a reduction may 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 the endogenous T cell receptor. Knockout of the cell surface expression of the endogenous T cell receptor (i.e., complete knockdown) can result from inactivation of the gene encoding the T cell receptor α constant region using an engineered meganuclease as described herein. The α constant region encoded by the T cell receptor α constant region gene is required for the assembly of the endogenous TCR complex on the cell surface. Thus, knockout of the T cell receptor α constant region gene using an engineered meganuclease as described herein results in knockout of the cell surface T cell receptor expression.

[0184] As used herein for both amino acid sequences and nucleic acid sequences, the terms "percent identity", "sequence identity", "percent similarity", "sequence similarity", etc. refer to a measure of the degree of similarity between two sequences based on a sequence alignment that maximizes the similarity between aligned amino acid residues or nucleotides, and which is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. A variety of algorithms and computer programs are available for determining 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. 25:3389-3402); Zhang et al. (2000), J. Comput. Biol. 7(1-2):203-14. As used herein, the percent similarity of two amino acid sequences is a score based on the following parameters of the BLASTp algorithm: word size = 3; gap open penalty = -11; gap extension penalty = -1; scoring matrix = BLOSUM62. As used herein, the percent similarity of two nucleic acid sequences is a score based on the following parameters for the BLASTn algorithm: word size = 11; gap open penalty = -5; gap extension penalty = -2; match reward = 1; mismatch penalty = -3.

[0185] As used herein, with respect to modifications of two proteins or amino acid sequences, the term "corresponds to" is used to indicate that a particular modification in a first protein is a replacement of the same amino acid residue in a modification of a second protein, and when the two proteins are subjected to a standard sequence alignment (e.g., using the BLASTp program), the position of the modified amino acid in the first protein corresponds to or aligns with the position of the modified amino acid in the second protein. Thus, if residues X and Y correspond to each other in a sequence alignment, a modification of residue "X" to amino acid "A" in the first protein will correspond to a modification of residue "Y" to amino acid "A" in the second protein, even though X and Y may be different numbers.

[0186] 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 homodimer or heterodimer meganuclease, or by a subunit of a single-stranded meganuclease.

[0187] As used herein, the term "hypervariable region" refers to a local sequence within a meganuclease monomer or subunit that contains amino acids with relatively high variability. The hypervariable region can contain about 50 - 60 contiguous residues, about 53 - 57 contiguous residues or preferably about 56 residues. In some embodiments, the residues of the hypervariable region can correspond to positions 24 - 79 or 215 - 270 of SEQ ID NO: 7 or 8. The hypervariable region can contain one or more residues that contact DNA bases in the recognition sequence and can be modified to alter the base preference of the monomer or subunit. The hypervariable region can also contain 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 for the DNA backbone and the target recognition sequence. In different embodiments of the present invention, the hypervariable region can contain 1 - 20 residues that exhibit variability and can be modified to affect base preference and / or DNA binding affinity (therein). In certain embodiments, the hypervariable region contains about 15 - 20 residues that exhibit variability and can be modified to affect base preference and / or DNA binding affinity.

[0188] In some embodiments, the variable residues within the 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 the 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 the 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 the 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.

[0189] In other embodiments, the variable residues within the 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.

[0190] As used herein, the terms "T cell receptor alpha gene" or "TCRα gene" are used interchangeably and refer to the locus in T cells that encodes the alpha subunit of the T cell receptor. Before or after rearrangement, the T cell receptor alpha can refer to NCBI Gene ID number 6955. After rearrangement, the T cell receptor alpha gene contains an endogenous promoter, rearranged V and J segments, an endogenous splice donor site, an intron, an endogenous splice acceptor site, and the T cell receptor alpha constant region locus (which contains the exons encoding the subunit).

[0191] As used herein, the term "T cell receptor alpha constant region" or "TCRα constant region" refers to the coding sequence of the T cell receptor alpha gene. The TCRα constant region contains the wild-type sequence as identified by NCBI Gen ID NO.28755 and its functional variants.

[0192] 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-stranded or double-stranded polynucleotides. A recombinant construct contains 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 contain regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source and arranged in a manner different from that found in nature. Such constructs can be used alone or in combination with a vector.

[0193] As used herein, a "vector" or "recombinant DNA vector" can be a construct that contains a replication system and sequences capable of transcribing and translating a polypeptide coding sequence in a given host cell. If a vector is used, the choice of vector depends on the method to be used for transforming the host cell, 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 the gene encoding the meganuclease of the present invention to a target cell. Those skilled in the art are well aware of the genetic elements that must be present on the vector for successful transformation, selection, and propagation of host cells containing any isolated nucleotide or nucleic acid sequence of the present invention.

[0194] As used herein, "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).

[0195] As used herein, a "polycistronic" mRNA refers to a single messenger RNA that contains two or more coding sequences (i.e., cistrons) and encodes more than one protein. A polycistronic mRNA can contain any element known in the art to permit 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.

[0196] As used herein, "human T cells" or "T cells" refers to T cells 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 under cell culture conditions but not immortalized, and T cells that have been immortalized and can be maintained indefinitely under cell culture conditions.

[0197] As used herein, "control" or "control cell" refers to a cell that provides a reference point for measuring genotypic or phenotypic changes in a cell that has been genetically modified. Control cells can include, for example: (a) wild-type cells, i.e., cells having the same genotype as the starting material used for genetic alteration of the genetically modified cell; (b) cells having the same genotype as the genetically modified cell, but that have been transformed with a null construct (i.e., a construct having no known effect on the trait of interest); or (c) cells that are genetically identical to the genetically modified cell, but that have not been exposed to conditions or stimuli or further genetic modifications that would induce expression of an altered genotype or phenotype.

[0198] As used herein, the term "treating" or "treating a subject" refers to administering to a subject having a disease a genetically modified T cell or population of genetically modified T cells of the present invention. For example, the subject can have a disease (e.g., cancer), and treatment can represent an immunotherapy for treating the disease. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or alleviating the disease state, and remitting or improving the prognosis. In some aspects, the genetically modified eukaryotic cells or population of genetically modified eukaryotic cells described herein are administered in the form of a pharmaceutical composition of the present invention during treatment.

[0199] The term "effective amount" or "therapeutically effective amount" refers to an amount 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 specific embodiments, an effective amount of the genetically modified T cells or population of genetically modified T cells of the present invention or the pharmaceutical composition disclosed herein alleviates at least one symptom of the disease in a subject. In those embodiments where the disease is cancer, an effective amount of the engineered meganuclease or pharmaceutical composition disclosed herein reduces the level of cancer proliferation or metastasis, causes a partial or complete response or remission of the cancer, or alleviates at least one cancer symptom in a subject.

[0200] As used herein, the term "cancer" is to be understood to encompass any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division leading to malignant growth or tumor.

[0201] As used herein, the term "carcinoma" refers to a malignant growth composed of epithelial cells.

[0202] As used herein, the term "leukemia" refers to a malignancy of the hematopoietic organs / system and is typically characterized by abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow.

[0203] As used herein, the term "sarcoma" refers to a tumor composed of substances similar to embryonic connective tissue and is typically composed of closely packed cells embedded in a fibrillar, heterogeneous, or homogeneous substance.

[0204] As used herein, the term "melanoma" refers to a tumor derived from the melanocyte system of the skin and other organs.

[0205] As used herein, the term "lymphoma" refers to a group of blood cell tumors that develop from lymphocytes.

[0206] As used herein, the term "blastoma" refers to a type of cancer caused by a malignancy in precursor cells or blast cells (immature or embryonic tissue).

[0207] As used herein, the recitation of a numerical range for a variable is intended to convey that the invention can be practiced with the variable equal to any value within that range. Thus, for a variable that is inherently discrete, the variable can be equal to any integer value within the numerical range, including the endpoints of the range. Similarly, for a variable that is inherently continuous, the variable can be equal to any real value within the numerical range, including the endpoints of the range. By way of example and not limitation, if a variable is inherently discrete, a variable described as having a value between 0 and 2 can take on the values 0, 1, or 2, and if the variable is inherently continuous, the variable can take on the values 0.0, 0.1, 0.01, 0.001, or any other real value ≥0 and ≤2.

[0208] 2.1 Invention Principle

[0209] The present invention is in part based on the discovery of optimized second-generation meganucleases that have improved properties compared to parental first-generation meganucleases, such as increased (i.e., enhanced) specificity and reduced off-target cleavage, reduced in-cell persistence after expression from mRNA, improved cellular properties when used with human T cells in vitro, and improved cellular properties when used in large-scale CAR T cell manufacturing processes.

[0210] 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 TCRα constant region gene. Cleavage at this recognition sequence can permit NHEJ at the cleavage site and disrupt the expression of the human T cell receptor alpha 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 permit direct homologous recombination of an exogenous nucleic acid sequence into the TCRα constant region gene. Such exogenous nucleic acid sequences can contain a sequence of interest, such as a sequence encoding a chimeric antigen receptor, an exogenous TCR receptor, or any other polypeptide of interest. Thus, the presently disclosed compositions and methods permit knockout of the endogenous T cell receptor (e.g., α / β T cell receptor) and expression of an exogenous nucleic acid sequence (e.g., chimeric antigen receptor or exogenous TCR). When administered to an allogeneic subject, such cells can exhibit reduced induction or no induction of graft-versus-host disease (GVHD).

[0211] 2.2 Optimization of a Megal nuclease that Recognizes and Cuts the TRC 1-2 Recognition Sequence within the T-Cell Receptor Alpha Constant Region Gene Nuclease

[0212] It is known in the art that site-specific nucleases can be used to create DNA breaks in the genome of living cells, and such DNA breaks can lead to permanent modification of the genome by homologous recombination of the cut target site with the same or highly homologous DNA sequence within the genome. Thus, in some embodiments, engineered recombinant meganucleases can be used to practice the present invention.

[0213] In certain embodiments, the nuclease used to practice the present invention is a single-stranded meganuclease. The single-stranded meganuclease comprises an N-terminal subunit and a C-terminal subunit linked by a linker peptide. Each of the two domains recognizes one half of the recognition sequence (i.e., a recognition 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 the meganuclease generates a pair of 3'-sided single-stranded overhangs of four base pairs.

[0214] The recombinant meganucleases of the present invention have been engineered to recognize and cleave the TRC 1-2 recognition sequence (SEQ ID NO: 5) within exon 1 of the TCRα constant region gene (SEQ ID NO: 3). The engineered meganucleases of the present invention comprise a first subunit and a second subunit, the first subunit comprising a first hypervariable (HVR1) region and the second subunit comprising a second hypervariable (HVR2) region. In addition, the first subunit binds to the first recognition half-site in the recognition sequence (i.e., the TRC1 half-site) and the second subunit binds to the second recognition half-site in the recognition sequence (i.e., the TRC2 half-site). In embodiments where the recombinant meganuclease is a single-stranded meganuclease, the first subunit and the second subunit can be oriented such that the first subunit comprising the HVR1 region and binding to the first half-site is positioned as the N-terminal subunit, and the second subunit comprising the HVR2 region and binding to 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 to the first half-site is positioned as the C-terminal subunit, while the second subunit comprising the HVR2 region and binding to 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.

[0215] Table 1. Exemplary engineered meganucleases that recognize and cleave the TRC 1-2 recognition sequence (SEQ ID NO: 5)

[0216]

[0217] * "HVR1%" and "HVR2%" represent the amino acid sequence identity between the HVR1 region and the HVR2 region of each meganuclease and the HVR1 region and the HVR2 region of the TRC 1-2x.87EE meganuclease, respectively.

[0218] In some embodiments, compared to the first-generation meganuclease TRC 1-2x.87EE, the currently disclosed engineered meganucleases exhibit at least one optimized property. Such optimized features include improved (i.e., increased) specificity (resulting in reduced off-target cleavage), reduced duration in cells after mRNA expression, and enhanced (i.e., increased) cleavage and modification efficiency of the TCRα constant region gene. Thus, in certain embodiments, when the currently disclosed engineered meganucleases are delivered to a population of eukaryotic cells, they are capable of generating a greater percentage of cells with cleavage and / or modification of the TCRα 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 that contain a cleavage and / or insertion / deletion ("indel") in the TCRα constant region gene. Any method known in the art can be used to measure the cleavage and / or modification of the TCRα constant region gene by the meganuclease, including the T7 endonuclease I assay, digital PCR, mismatch detection assay, mismatch cleavage assay, high-resolution melting analysis (HRMA), heteroduplex mobility assay, sequencing, and fluorescence PCR capillary gel electrophoresis (see, e.g., Zischewski et al. (2017) Biotechnology Advances 35(1):95-104, the entire content of which is incorporated herein by reference).

[0219] In certain embodiments, compared to the first-generation TRC 1-2x.87EE meganuclease, the currently disclosed engineered meganucleases exhibit a reduced duration in cells, particularly when introduced as mRNA. Any method known in the art can be used to measure the persistence of intracellular mRNA or protein, including but not limited to RT-PCR, RNA blotting, nuclease protection assay, in situ hybridization, immunocytochemistry, immunoblotting, and immunoprecipitation.

[0220] 2.3 Methods for Delivering and Expressing the Optimized Megal nuclease

[0221] The present invention provides methods for generating genetically modified T cells and populations thereof using engineered meganucleases that recognize and cleave recognition sequences present in the human TCRα 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, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure, any number of T cell lines available in the art can be used. In some embodiments of the present disclosure, T cells are obtained from blood units 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.

[0222] The modified T cell receptor α gene contains an exogenous sequence of interest inserted into the first exon (i.e., the target exon) of the TCRα constant region gene by double-strand cleavage by the presently disclosed engineered meganucleases. The cleavage sites generated by such meganucleases can permit direct homologous recombination of the exogenous sequence of interest into the target exon.

[0223] As used herein, the term “exogenous” or “heterologous” with respect to a nucleotide sequence means a sequence that is either purely synthetic, derived from a foreign source, or, if from the same source, has been substantially altered in composition and / or genomic locus from its native form through deliberate human intervention.

[0224] In various embodiments, the exogenous sequence of interest can contain a coding sequence for a protein of interest. It is contemplated that the coding sequence can be for any protein of interest.

[0225] In certain embodiments, the exogenous sequence of interest contains a nucleic acid sequence encoding a chimeric antigen receptor (CAR). Generally, the CARs of the present disclosure will comprise at least an extracellular domain and an intracellular domain. In some embodiments, the extracellular domain contains a target-specific binding element, also referred to as a ligand-binding domain or moiety. In some embodiments, the intracellular domain or cytoplasmic domain contains at least one co-stimulatory domain and one or more signaling domains, such as CD3ζ.

[0226] In some embodiments, the CARs useful in the present invention comprise extracellular target-specific binding elements, also referred to as ligand-binding domains or moieties. The choice of ligand-binding domain depends on the type and number of ligands that define the target cell surface. For example, a ligand-binding domain can be selected to recognize a ligand that serves as a cell surface marker associated with a particular disease state on the target cell. Thus, examples of cell surface markers that can serve as ligands for ligand-binding domains in CARs can include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells. In some embodiments, the CARs can be engineered to target tumor-specific target antigens by engineering a desired ligand-binding moiety that specifically binds an antigen on the 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 such as cancer.

[0227] 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, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipid, glioma-associated antigen, human chorionic gonadotropin beta, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, survivin and telomerase, prostate cancer tumor antigen 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin-like growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stroma antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and A1 domain of tenascin C (TnC A1) and fibroblast activation protein (fap); lineage-specific 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, TNFRSF17), CS1; or virus-specific surface antigens such as HIV-specific antigens (e.g., HIV gp120), EBV-specific antigens, CMV-specific antigens, HPV-specific antigens (e.g., E6 or E7 oncoproteins), Lassa virus-specific antigens, influenza virus-specific antigens, and any derivatives or variants of these surface markers. In certain embodiments of the present disclosure, the ligand-binding domain is specific for CD19.

[0228] 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 the autoantigen-specific B cell receptor on B lymphocytes, thereby guiding T cell-specific targeting and killing autoreactive B lymphocytes in antibody-mediated autoimmune diseases. Such a CAR can be referred to as a chimeric autoantibody receptor (CAAR).

[0229] In some embodiments, the extracellular domain of the chimeric antigen receptor may comprise a naturally occurring ligand of the antigen of interest or a fragment of a naturally occurring ligand that retains the ability to bind the antigen of interest.

[0230] In some embodiments, the CAR comprises a transmembrane domain that connects the extracellular ligand-binding domain or autoantigen to the intracellular signaling and co-stimulatory domains via 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 α, β, γ, or ζ polypeptide that constitutes the CD3 complex), the IL2 receptor p55 (α chain), p75 (β chain), or γ chain, a subunit chain of the Fc receptor (e.g., Fcγ receptor III), or a CD protein (e.g., CD8α chain). Alternatively, the transmembrane domain can be synthetic and can mainly comprise hydrophobic residues such as leucine and valine. In a particular embodiment, the transmembrane domain is a CD8α transmembrane polypeptide.

[0231] The hinge region refers to any oligomer or polypeptide having the function of connecting the transmembrane domain to the extracellular ligand-binding domain. For example, the hinge region can comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region can be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or all or part of the antibody constant region. Alternatively, the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a fully synthetic hinge sequence. In a particular embodiment, the hinge domain can comprise a portion of the human CD8α chain, the FcγRIIIa receptor, or IgG1.

[0232] The intracellular signaling domain of the 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 the specialized functions of a cell. The effector functions of a T cell can be, for example, cytolytic activity or helper activity, including the secretion of cytokines. The intracellular signaling domain (such as CD3ζ) can provide an activation signal to the cell in response to the binding of the extracellular domain. As discussed, the activation signal can induce effector functions of the cell, such as cytolytic activity or cytokine secretion.

[0233] The intracellular domain of the CAR can include one or more intracellular co-stimulatory domains that transmit co-stimulatory signals after extracellular domain binding to promote cell proliferation, cell survival, and / or cytokine secretion. Such intracellular co-stimulatory 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 ligands that specifically bind to CD83.

[0234] The CAR can be specific for any type of cancer cell. Such cancers can include but are not limited to epithelial cancer, lymphoma, sarcoma, blastoma, leukemia, cancers 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 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 lymphoma, and multiple myeloma.

[0235] The target sequence can further encode an exogenous T cell receptor (TCR). Such an exogenous T cell receptor can comprise α and β chains, or optionally can comprise γ and δ chains. The exogenous TCRs useful in the present invention can be specific for any target antigen or epitope.

[0236] In other embodiments, the target sequence can encode a wild-type or modified form of an endogenous target gene.

[0237] The target sequence can contain elements or peptides known in the art to allow the 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 certain embodiments, such elements in the exogenous target sequence can be located 5' upstream or 3' downstream of the nucleic acid sequence encoding the target protein (such as the CAR).

[0238] The exogenous target sequence described herein may further comprise additional control sequences. For example, the target sequence may comprise a homologous recombination enhancer sequence, a Kozak sequence, a polyadenylation sequence, a transcription termination sequence, a selection marker sequence (such as an antibiotic resistance gene), an origin of replication, etc. The target sequence described herein may also comprise at least one nuclear localization signal. Examples of nuclear localization signals are known in the art (see, for example, Lange et al., J. Biol. Chem., 2007, 282:5101-5105).

[0239] The engineered meganucleases of the invention can be delivered into cells 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 PCR product) or RNA (e.g., mRNA). For embodiments in which the engineered meganuclease coding 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 present invention include constitutive promoters (such as the cytomegalovirus immediate 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))) and inducible promoters (such as the tetracycline-inducible promoter ((Dingermann et al. (1992), Mol Cell Biol. 12(9):4038-45))). The engineered meganucleases of the invention can also be operably linked to a synthetic promoter. Synthetic promoters can include, but are not limited to, the JeT promoter (WO 2002 / 012514).

[0240] In some embodiments, mRNA encoding the engineered meganuclease is delivered to the cells because this reduces the likelihood of the gene encoding the engineered meganuclease integrating into the cell genome. Such mRNA encoding the engineered meganuclease can be produced using methods known in the art such as in vitro transcription. In some embodiments, the mRNA uses 7-methylguanosine, an anti-reverse cap analog (ARCA) (US 7,074,596), Analogs such as Cap1 analogs (Trilink, San Diego, CA) are used to 5'-cap, or enzymatic capping is carried out using vaccinia capping enzyme or in a similar manner. In some embodiments, the mRNA can be polyadenylated. The mRNA can contain various 5' and 3' untranslated sequence elements to enhance the expression of the encoded engineered meganuclease and / or the stability of the mRNA itself. Such elements can include, for example, post-translational regulatory elements (such as the woodchuck hepatitis virus post-translational regulatory element). The mRNA can contain nucleoside analogs or naturally occurring nucleosides, such as pseudouridine, 5-methylcytidine, N6-methyladenosine, 5-methyluridine, or 2-thiouridine. Additional nucleoside analogs include, for example, those described in US 8,278,036.

[0241] In certain embodiments, the mRNA encoding the engineered meganuclease of the present invention can be a polycistronic mRNA encoding two or more meganucleases that are co-expressed 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 a second recognition sequence located in a second gene, thereby generating cleavage sites in both genes. The polycistronic mRNA can contain any element known in the art to allow translation of two or more genes (i.e., cistrons) from the same mRNA molecule, and such elements include, but are not limited to, IRES elements, T2A elements, P2A elements, E2A elements, and F2A elements.

[0242] In another specific embodiment, a single-stranded DNA template can be used to introduce the nucleic acid encoding the engineered meganuclease of the present invention into a cell. The single-stranded DNA can also contain 5' and / or 3' AAV inverted terminal repeats (ITRs) upstream and / or downstream of the sequence encoding the engineered meganuclease. In other embodiments, the single-stranded DNA can further contain 5' and / or 3' homologous arms upstream and / or downstream of the sequence encoding the engineered meganuclease.

[0243] In another specific embodiment, a linearized DNA template can be used to introduce the gene encoding the meganuclease of the present invention into a cell. In some examples, plasmid DNA encoding the meganuclease can be digested with one or more restriction enzymes such that the circular plasmid DNA is linearized before being introduced into the cell.

[0244] Purified meganuclease proteins can be delivered into cells by a variety of different mechanisms known in the art, including those further detailed below, to cleave genomic DNA, which allows for homologous recombination or non-homologous end joining with the target sequence at the cleavage site.

[0245] In some embodiments, the meganuclease protein or DNA / mRNA encoding a 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), the 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 a meganuclease is covalently or non-covalently conjugated to an antibody that recognizes a specific cell surface receptor expressed on the target cell, thereby enabling 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 the natural ligand (or a portion of the natural ligand) of 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).

[0246] In some embodiments, the meganuclease protein or DNA / mRNA encoding a meganuclease is covalently or non-covalently conjugated to or encapsulated within nanoparticles using methods known in the art (Sharma, et al. (2014) Biomed Res Int. 2014). Nanoparticles are nanoscale delivery systems with a length scale <1 µm, preferably <100 nm. Such nanoparticles can be designed using a core composed of metal, lipid, polymer, or biopolymer, 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 cleavage of the target recognition sequence. The surface of such nanoparticles can be further modified with polymers or lipids (such as chitosan, cationic polymers, or cationic lipids) to form core-shell nanoparticles whose surface confers additional functions to enhance delivery and cellular uptake of the payload (Jian et al. (2012) Biomaterials. 33(30):7621-30). Nanoparticles can additionally advantageously be conjugated to targeting molecules to direct the nanoparticles to appropriate cell types 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.

[0247] In some embodiments, the meganuclease protein or DNA / mRNA encoding a meganuclease is encapsulated within liposomes or complexed using cationic lipids (see, for example, 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 cationic liposome (lipoplex) formulations can protect the payload from degradation and promote cellular uptake and delivery efficiency by fusing with the cell membrane of the target cell and / or disrupting the cell membrane of the target cell.

[0248] In some embodiments, the meganuclease protein or DNA / mRNA encoding a meganuclease is encapsulated within a polymeric scaffold (such as PLGA) or complexed using cationic polymers (such as PEI, PLL) (Tamboli et al. (2011) TherDeliv. 2(4):523-536). The polymeric carrier can be designed to provide a tunable drug release rate 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.

[0249] In some embodiments, the meganuclease protein or DNA / mRNA encoding a recombinant meganuclease is conjugated to an amphiphile that self-assembles into micelles (Tong et al. (2007) J Gene Med. 9(11):956-66). The polymeric micelles can include a micelle shell formed from a hydrophilic polymer (such as polyethylene glycol), which can prevent aggregation, shield charge interactions, and reduce non-specific interactions.

[0250] In some embodiments, the meganuclease protein or DNA / mRNA encoding a meganuclease is formulated into 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, but is not limited to, any oil-in-water, water-in-oil, water-in-oil-in-water or oil-in-water-in-oil dispersion or droplet (including lipid structures), which can form as a result of the hydrophobic force that drives non-polar residues (such as long hydrocarbon chains) away from water and polar head groups towards water when a water-insoluble phase is mixed with an aqueous phase. These other lipid structures include, but are not limited to, monolayers, paucilamellar and multilamellar lipid vesicles, micelles, and lamellar phases. Emulsions consist of an aqueous phase and a lipophilic phase (usually containing oil and organic solvents). Emulsions also often contain 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.

[0251] In some embodiments, meganuclease proteins or DNA / mRNA encoding meganucleases are covalently linked or non-covalently associated with multifunctional polymers, 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). Dendrimer generation can control payload capacity and size and can provide high drug payload capacity. Moreover, the display of multiple surface groups can be utilized to improve stability, reduce non-specific interactions, and enhance cell-specific targeting and drug release.

[0252] In some embodiments, viral vectors are used to deliver genes encoding meganucleases. Such vectors are known in the art and include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated virus (AAV) vectors (reviewed in Vannucci et al. 2013 New Microbiol. 36:1-22). Recombinant AAV vectors useful in the present invention can have any serotype that permits transduction of the virus into cells and insertion of the nuclease gene into the cell genome. In certain embodiments, the recombinant AAV vector has a serotype of AAV2 or AAV6. AAV vectors 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).

[0253] If meganuclease genes are delivered in DNA form (e.g., plasmid) and / or via viral vectors (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 a viral vector (e.g., the LTR of a lentiviral vector) or the well-known cytomegalovirus or SV40 virus early promoter. In a preferred embodiment, the meganuclease gene is operably linked to a promoter that preferentially drives gene expression in target cells (e.g., T cells).

[0254] The present invention is further directed to introducing an exogenous target sequence into the T cell receptor alpha constant region gene at the TRC 1-2 recognition sequence. In some embodiments, the exogenous target sequence comprises a 5' homology arm and a 3' homology arm flanking an element of the insert. Such homology arms have sequence homology with the corresponding sequences 5' upstream and 3' downstream of the nuclease recognition sequence at which the cleavage site is generated. Generally, the length of the homology arms can be at least 50 base pairs, 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 higher sequence homology with their corresponding sequences in the genome.

[0255] The exogenous target sequence of the present invention can be introduced into cells by any of the methods discussed above. In certain embodiments, the exogenous target sequence is introduced by a viral vector, such as a lentivirus, retrovirus, adenovirus, or preferably a recombinant AAV vector. The recombinant AAV vector that can be used to introduce exogenous nucleic acids can have any serotype that allows transduction of the virus into cells and insertion of the exogenous nucleic acid sequence into the cell genome. In certain embodiments, the recombinant AAV vector has a 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 DNA in the host cell.

[0256] In another particular embodiment, a single-stranded DNA template can be used to introduce the exogenous target sequence into cells. The single-stranded DNA can comprise the exogenous target sequence and, in a preferred embodiment, 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) upstream of the 5' of the 5' homology arm and a 3' AAV ITR sequence downstream of the 3' of the 3' homology arm.

[0257] In another particular embodiment, the gene encoding the engineered nuclease of the present invention and / or the exogenous target sequence of the present invention can be introduced into cells by transfection with a linearized DNA template. In some examples, plasmid DNA can be digested with one or more restriction enzymes such that the circular plasmid DNA is linearized before transfection into cells.

[0258] The T cells modified by the present invention may need to be activated before meganuclease and / or exogenous target sequence introduction. For example, the T cells can be contacted with anti-CD3 and anti-CD28 antibodies that are soluble or conjugated to a support (i.e., beads) for a time sufficient to activate the cells.

[0259] The genetically modified cells of the present invention can be further modified to express one or more inducible suicide genes, the induction of which causes cell death and permits the 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 having 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 encoding a product that causes cell death either by itself or in the presence of other compounds. Representative examples of such suicide genes are genes encoding thymidine kinase of herpes simplex virus. Additional examples are genes encoding thymidine kinase of varicella zoster virus and bacterial cytosine deaminase genes that can convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil. As non-limiting examples, suicide genes also include genes encoding caspase-9, caspase-8 or cytosine deaminase. In some examples, a specific chemical inducer of dimerization (CID) can be used to activate caspase-9. The suicide gene can also encode a polypeptide expressed on the cell surface that renders the cell sensitive to therapeutic and / or cytotoxic monoclonal antibodies. In further examples, the suicide gene can encode a recombinant antigen polypeptide comprising an antigenic motif recognized by the anti-CD20 mAb rituximab and an epitope that permits the selection of cells expressing the suicide gene. See, for example, the RQR8 polypeptide described in WO2013153391, which comprises two rituximab-binding epitopes and a QBEnd10-binding epitope. For such a gene, rituximab can be administered to a subject when needed to induce cell depletion. In further examples, the suicide gene can include a QBEnd10-binding epitope expressed in combination with a truncated EGFR polypeptide.

[0260] Eukaryotic cells modified by the methods and compositions described herein can have reduced expression of endogenous T cell receptors (i.e., α / β T cell receptors), and can optionally further express a protein of interest (e.g., CAR). Accordingly, the present invention further provides a population of eukaryotic cells that express a protein of interest and do not express endogenous T cell receptors (e.g., α / β T cell receptors). For example, the population can include a plurality of genetically modified eukaryotic cells of the present invention that express CAR (i.e., CAR+) or an exogenous T cell receptor (i.e., exoTCR+), and have reduced expression of endogenous T cell receptors (i.e., TCR-). In various embodiments of the present invention, 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 a particular instance, 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 contained in the population can be TCR- and CAR+.

[0261] In some embodiments, when the presently disclosed engineered meganucleases are introduced into a cell population, a higher percentage of TCR- and CAR+ cell populations are produced compared to when the first generation TCR 1-2x.87EE meganuclease is introduced into the cell population.

[0262] In addition, cells genetically modified with the presently disclosed engineered meganucleases exhibit improved characteristics compared to cells genetically modified with the TRC1-2x.87EE meganuclease over a wide range, including reduced off-target cleavage and its effects, reduced persistence of the meganuclease in the cells, enhanced (i.e., increased) CAR T expansion, and lower differentiation. Additionally, cell populations 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 low-differentiated cells compared to those cell populations into which the TRC1-2x.87EE meganuclease (or nucleic acid encoding the same) has been introduced. In certain embodiments, cell populations into which the presently disclosed engineered meganucleases have been introduced exhibit a higher percentage of central memory T cells (e.g., those expressing CD45RO, CCR7, and CD62L) compared to cell populations into which the first-generation TRC1-2x.87EE meganuclease has been introduced.

[0263] 2.4 Pharmaceutical Compositions

[0264] In some embodiments, the present invention provides a pharmaceutical composition comprising a genetically modified eukaryotic cell or a population of genetically modified eukaryotic cells of the present invention, 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 manufacturing a pharmaceutical preparation according to the present invention, the cells are generally admixed 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 must not be harmful to the subject. In some embodiments, the pharmaceutical compositions of the present invention may further comprise one or more additional agents useful for treating a disease of the subject. In further embodiments, the pharmaceutical compositions of the present invention may further comprise biomolecules, such as cytokines (e.g., IL-2, IL-7, IL-15, and / or IL-21), which promote in vivo cell proliferation and engraftment of genetically modified T cells. A pharmaceutical composition comprising a genetically modified eukaryotic cell of the present invention can be administered in the same composition with an additional agent or biomolecule, or can be co-administered in separate compositions.

[0265] The present disclosure also provides the genetically modified cells or populations thereof described herein for use as a medicament. The present disclosure further provides the use of the genetically modified cells or populations thereof described herein in the preparation of a medicament for treating a disease in a subject in need thereof. In one such aspect, the medicament can be used for cancer immunotherapy in a subject in need thereof.

[0266] It is known that cells into which the meganucleases disclosed in the present invention are introduced can reduce off-target cleavage, reduce the duration of meganucleases in cells, improve the disruption efficiency of the TCRα 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, compared to the administration of a pharmaceutical composition comprising cells genetically modified with the TRC1-2x.87EE meganuclease, the presently disclosed pharmaceutical composition comprising genetically modified cells also has improved efficacy in treating diseases (such as cancer) when administered to a subject in need thereof.

[0267] In some embodiments, compared to the introduction of the first-generation TCR 1-2x.87EE meganuclease into a cell population, the presently disclosed engineered meganucleases produce a higher percentage of TCR- and CAR+ cell populations when introduced into a cell population.

[0268] Furthermore, compared to cells that have been genetically modified with the TRC1-2x.87EE meganuclease, cells that have been genetically modified with the presently disclosed engineered meganucleases exhibit improved characteristics, including reduced off-target cleavage and its effects, reduced duration of meganucleases in cells, enhanced (i.e., increased) CAR T expansion, and lower cell differentiation. Additionally, compared to a cell population into which the TRC1-2x.87EE meganuclease (or nucleic acid encoding the same) has been introduced, a cell population into 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. In certain embodiments, compared to a cell population into which the parental TRC1-2x.87EE meganuclease has been introduced, a cell population into which the presently disclosed engineered meganuclease has been introduced exhibits a greater percentage of central memory T cells (such as those expressing CD45RO, CCR7, and CD62L).

[0269] The pharmaceutical compositions of the present invention can be used to treat any disease state that can be targeted by adoptive T cell immunotherapy. In certain embodiments, the pharmaceutical compositions and medicaments of the present invention 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 epithelial cancers, 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, liver cancer, gastric cancer, 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 area, cancer of the stomach, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers (including asbestos-induced cancers), multiple myeloma, Hodgkin lymphoma, non-Hodgkin 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 the foregoing 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 lymphoma, multiple myeloma, and B cell non-Hodgkin lymphoma.

[0270] In some of these embodiments in which cancer is treated with the presently disclosed genetically modified cells or populations thereof, an additional therapeutic agent, such as radiation, surgery, or chemotherapeutic agent, is further administered to a subject to whom the genetically modified cells or populations thereof have been administered.

[0271] The present invention further provides a population of genetically modified cells comprising a plurality of the genetically modified cells described herein, wherein the cells comprise an exogenous nucleic acid molecule encoding a sequence of interest in their genome, wherein the exogenous nucleic acid molecule is inserted into the T cell receptor alpha constant region gene, and wherein the cell surface expression of the endogenous TCR is reduced. Thus, in various embodiments of the present invention, a population of genetically modified cells is provided, 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 the genetically modified cells described herein. In other embodiments of the present invention, a population of genetically modified cells is provided, 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 are the genetically modified cells described herein that further express a chimeric antigen receptor.

[0272] 2.5 Methods of Administration of Genetically Modified Cells

[0273] Another aspect disclosed herein is administering an effective amount of the genetically modified eukaryotic cells or a population thereof of the present disclosure to a subject in need thereof. In certain embodiments, the pharmaceutical composition described herein is administered to a subject in need thereof. For example, an effective amount of a population of cells can be administered to a subject suffering from a disease. In certain embodiments, the disease can be cancer, and the administration of the genetically modified eukaryotic cells of the present invention represents immunotherapy. The administered cells are capable of reducing the proliferation of target cells in the recipient, reducing their number or killing the target cells. Different from antibody therapy, the genetically modified eukaryotic cells of the present disclosure are capable of replicating and expanding in vivo, resulting in long-term persistence, which can lead to continuous control of the disease.

[0274] Examples of possible routes of administration include parenteral (e.g., intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC) or infusion) administration. In addition, it can be administered by continuous infusion or by single or multiple boluses. In certain 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.

[0275] In some embodiments, for the purpose of treating cancer, the genetically modified eukaryotic cells or populations thereof of the present disclosure target tumor antigens. Such cancers can include, but are not limited to, epithelial cancers, lymphomas, sarcomas, blastomas, leukemias, cancers 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 lymphoma. In certain 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 combinations 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 epithelial cancers, 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, liver cancer, gastric cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, cancer of the stomach, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem gliomas, pituitary adenomas, Kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers (including asbestos-induced cancers), multiple myeloma, Hodgkin lymphoma, non-Hodgkin 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 the foregoing 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 lymphoma, multiple myeloma, and B-cell non-Hodgkin lymphoma.

[0276] When indicating an “effective amount” or “therapeutically effective amount”, it can be determined by a physician based on individual differences in age, body weight, tumor size (if present), degree of infection or metastasis, and the condition of the patient (subject). In some embodiments, the pharmaceutical composition comprising the genetically modified cells or a population thereof described herein is administered at a dose of 10 4 to 10 9 cells / kg body weight, including all integer values within those ranges. In additional 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). By monitoring the signs of the patient's disease and adjusting the treatment accordingly, one of ordinary skill in the medical art can readily determine the optimal dose and treatment regimen for a particular patient.

[0277] In some embodiments, administration of the genetically modified eukaryotic cells or a population thereof of the present disclosure alleviates at least one symptom of the target disease or disorder. For example, administration of the genetically modified T cells or a population thereof of the present disclosure can alleviate at least one symptom of cancer. The symptoms of cancer are well known in the art and can be determined by known techniques.

[0278] 2.6 Methods for Generating Recombinant Viral Vectors

[0279] In some embodiments, the present invention provides viral vectors (e.g., recombinant AAV vectors) for the methods of the present invention. Recombinant AAV vectors are typically produced in mammalian cell lines such as HEK-293. Since the viral cap and rep genes are removed from the vector to prevent self-replication, creating space for the therapeutic gene to be delivered (e.g., a meganuclease gene), 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, where 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 sequence to be packaged into the virus. The viral particles containing the genome (ITRs and the intervening gene of interest) encapsulated 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 cells, tissues, or organisms (e.g., a human patient).

[0280] Since recombinant AAV particles are typically produced (manufactured) in cells, precautions must be taken in practicing the present invention to ensure that engineered meganucleases are not expressed in the packaging cells. Since the viral genome of the present invention can contain recognition sequences for meganucleases, any meganucleases expressed in the packaging cell line may be able to cleave the viral genome before it is packaged into viral particles. This would result in reduced packaging efficiency and / or packaging of fragmented genomes. Several methods can be used to prevent the expression of meganucleases in packaging cells, including:

[0281] Meganucleases can be placed under the control of tissue-specific promoters that are inactive in packaging cells. For example, if developing a viral vector 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 α1-antitrypsin (e.g., Pa1AT), and hemopexin (e.g., Phpx) (Kramer et al., (2003) Mol. Therapy 7:375-85), a hybrid liver-specific promoter (the hepatic locus control region from the ApoE gene (ApoE-HCR) and the liver-specific α1-antitrypsin promoter), the human thyroxine-binding globulin (TBG) promoter, and the apolipoprotein A-II promoter. Examples of eye-specific promoters include the rhodopsin and corneal epithelial-specific K12 promoter (Martin et al. (2002) Methods (28):267-75) (Tong et al., (2007) J GeneMed, 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 present invention, are not expected to produce significant levels of meganuclease gene expression in packaging cells. Similarly, the viral vectors of the present invention contemplate the use of other cell lines and the use of incompatible tissue-specific promoters (i.e., the well-known HeLa cell line (human epithelial cells) and 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-regulating APOM (liver), ADPRHL1 (heart), and monogenic malformation syndrome TP73L (muscle). (Jacox et al., (2010), PLoS One v.5(8):e12274).

[0282] Alternatively, the vector can be packaged in cells from a different species that are unlikely to express a broad range of nucleases. For example, mammalian promoters that are inactive in non-mammalian packaging cells (such as the well-known cytomegalovirus or SV40 virus early promoters) can be used to produce viral particles in microbial, insect, or plant cells. In a preferred embodiment, as described by Gao et al. (Gao et al. (2007), J. Biotechnol. 131(2):138-43), a baculovirus system is used to produce viral particles in insect cells. A broad range of nucleases 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 mammalian introns, such as the human growth hormone (HGH) intron or the SV40 large T antigen intron, into the coding sequence of a broad range of nucleases. Since these introns cannot be efficiently spliced from the pre-mRNA transcript in insect cells, the insect cells will not express a functional broad range of nucleases and will package the full-length genome. In contrast, mammalian cells that receive the produced recombinant AAV particles will correctly splice the pre-mRNA and express a functional broad range of nuclease protein. Haifeng Chen reported the use of the HGH and SV40 large T antigen introns to attenuate the expression of the toxic proteins Bacillus ribonuclease 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).

[0283] Meganuclease genes can be operably linked to inducible promoters 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-inducible transcription factors (variants of the Tet repressor and the ecdysone receptor, respectively), which activate transcription in response to small molecule activators (doxycycline or ecdysone, respectively). Implementing the present invention 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. This latter step is necessary because, without the simultaneous delivery of the transcriptional activator to the target cells, meganucleases will not be expressed in the target cells or tissues following recombinant AAV delivery. The transcriptional activator then induces meganuclease gene expression only in cells or tissues treated with the cognate small molecule activator. This method is advantageous because it enables the 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) presents a drawback to this method.

[0284] In another preferred embodiment, recombinant AAV particles are produced in mammalian cell lines that express a transcriptional repressor that blocks the expression of the meg nuclease. Transcriptional repressors are known in the art and include Tet repressor, Lac repressor, Cro repressor, and Lambda repressor. In the absence of the cognate hormone ligand, many nuclear hormone receptors such as the ecdysone receptor can also act as transcriptional repressors. To practice the invention, packaging cells are transfected / transduced with a vector encoding the transcriptional repressor, and the meg nuclease 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 a number of positions. It can be encoded on a separate vector; it can be incorporated into the packaging vector outside of the ITR sequences; it can be incorporated into the cap / rep vector or an adenovirus helper vector; or it can be stably integrated into the genome of the packaging cells such that it is expressed constitutively. Methods for 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 the operator of 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 non-human transcriptional repressors ensures that transcription of the meg nuclease 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 vector.

[0285] 2.7 Engineered Nuclease Variants

[0286] Embodiments of the invention include the engineered nucleases and variants thereof described herein. Other embodiments of the invention include polynucleotides comprising a nucleic acid sequence encoding a nuclease described herein, and variants of such polynucleotides.

[0287] As used herein, "variant" is intended to denote 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 in 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 parental sequence from which the 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; namely, the ability to recognize and cleave the TRC 1-2 recognition sequence (SEQ ID NO:5) found in the human T cell receptor α 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) modification efficiency of the TCRα constant region gene. Such variants can be generated, for example, by human manipulation. Biological activity variants of the native polypeptides of the embodiments (e.g., SEQ ID NOs: 7 and 8), or biological activity variants 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 the sequence alignment procedures and parameters described elsewhere herein. Biological activity variants of the polypeptides or subunits of the embodiments may differ from the 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 residue.

[0288] The polypeptides of the embodiments can be modified in various ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulation are well known in the art. For example, amino acid sequence variants can be prepared by mutagenesis of DNA. Methods of 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 the references cited therein. Guidelines for appropriate amino acid substitutions that do not affect the biological activity of the protein of interest 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 for another with similar properties, may be optimal.

[0289] In some embodiments, the engineered meganucleases of the invention can comprise variants of the HVR1 and HVR2 regions disclosed herein. The parental HVR regions 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 higher sequence identity to the amino acid sequence corresponding to residues 24-79 or residues 215-270 of the exemplified engineered meganucleases herein, such that the variant HVR region retains the biological activity of the engineered meganuclease (i.e., binds and cleaves the recognition sequence). Additionally, in some embodiments of the invention, the variant HVR1 region or the variant HVR2 region can comprise residues corresponding to the amino acid residues present at specific positions within the parental HVR. In such cases, "corresponding to" means that the amino acid residue in the variant HVR is the same amino acid residue (i.e., the identical residue alone) that is present in the parental HVR sequence at the same relative position (i.e., relative to the remaining amino acids of the parental sequence). For example, if the parental HVR sequence comprises a serine residue at position 26, then the variant HVR that "comprises a residue corresponding to" residue 26 will also comprise a serine at the position corresponding to (i.e., relative to) parental position 26.

[0290] In certain embodiments, the engineered meganucleases of the invention 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 higher sequence identity to the amino acid sequence corresponding to residues 215-270 of SEQ ID NO:7.

[0291] In certain embodiments, the engineered meganucleases of the invention 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 higher sequence identity to the amino acid sequence corresponding to residues 24-79 of SEQ ID NO:7.

[0292] In some embodiments, the engineered meganucleases of the invention comprise an 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 higher sequence identity to the amino acid sequence corresponding to residues 24 - 79 of SEQ ID NO:8.

[0293] In certain embodiments, the engineered meganucleases of the invention comprise an HVR1 region having at least 97% sequence identity to the amino acid sequence corresponding to residues 215 - 270 of SEQ ID NO:7 and an HVR2 region having at least 81% sequence identity to the amino acid sequence corresponding to residues 24 - 79 of SEQ ID NO:7.

[0294] In other specific embodiments, the engineered meganucleases of the invention comprise an HVR1 region having the amino acid sequence corresponding to residues 215 - 270 of SEQ ID NO:8 and an HVR2 region having at least 86% sequence identity to the amino acid sequence corresponding to residues 24 - 79 of SEQ ID NO:8.

[0295] Numerous amino acid modifications to the DNA recognition domain of wild - type I - CreI meganuclease have been previously identified (e.g., US 8,021,867), which alone or in combination have produced recombinant meganucleases with altered specificity at a single base within the DNA recognition sequence half - site, such that the resulting rationally designed meganucleases have a different half - site specificity from the wild - type enzyme. Table 2 provides potential substitutions that can be made at each half - site position (-1 to -9) of the recognition half - site in an engineered meganuclease monomer or subunit to enhance specificity.

[0296] Table 2.

[0297]

[0298]

[0299] Bold is the wild - type contact residue and does not constitute a "modification" as used herein. An asterisk indicates that the residue contacts the base on the antisense strand.

[0300] Certain modifications can be made in engineered meganuclease monomers or subunits to modulate DNA binding affinity and / or activity. For example, the engineered meganuclease monomers or subunits described herein can contain G, S, or A at the residue corresponding to position 19 of I-CreI or SEQ ID NO:7 or 8 (WO 2009001159), Y, R, K, or D at the residue corresponding to position 66 of I-CreI or SEQ ID NO:7 or 8, and / or E, Q, or K at the residue corresponding to position 80 of I-CreI or SEQ ID NO:7 or 8 (US8021867).

[0301] For polynucleotides, "variant" includes the deletion and / or addition of one or more nucleotides at one or more sites within a native polynucleotide. Those skilled in the art will recognize that variants of the nucleic acids of this embodiment are constructed such that the open reading frame is maintained. For polynucleotides, conservative variants include those sequences that encode the amino acid sequence of one of the polypeptides of the embodiment due to the degeneracy of the genetic code. Variant polynucleotides include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but still encoding a recombinant nuclease of the embodiment. Generally, variants of a particular polynucleotide of the embodiment 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 higher sequence identity with the particular polynucleotide, as determined by the sequence alignment programs and parameters described elsewhere herein. Variants of a particular polynucleotide of the embodiment (i.e., the reference polynucleotide) can also be evaluated by comparing the percent sequence identity between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide.

[0302] It is expected that deletions, insertions, and substitutions in the variant protein sequences encompassed herein will not produce a radical change in the properties of the polypeptide. However, when it is difficult to predict the exact effect of a substitution, deletion, or insertion prior to doing so, those skilled in the art will understand that the effect is evaluated by screening 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 α constant region gene (SEQ ID NO:3).

[0303] Examples

[0304] The present invention 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 within the scope of the claims following the examples below.

[0305] Example 1

[0306] Characterization of Megal nucleases Specific for the TRC 1-2 Recognition Sequence

[0307] 1. Megal nucleases that Recognize and Cut the TRC 1-2 Recognition Sequence

[0308] Second-generation TRC1-2 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) present in the human T cell receptor α constant region. Each of these second-generation meganucleases contains an N-terminal nuclease localization 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 contain a 56-base pair hypervariable region, designated HVR1 and HVR2, respectively.

[0309] The HVR1 region of each TRC1-binding subunit consists of residues 215-270 of SEQ ID NOs: 7 and 8. The TRC1-binding subunits of TRC 1-2L.1592 and TRC 1-2L.1775 are identical to each other outside the HVR1 region. The HVR1 region of each TRC 1-2 meganuclease contains modifications relative to the wild-type I-CreI sequence (SEQ ID NO: 1) at positions 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 266, and 268. Although not modified relative to wild-type I-CreI, the arginine residue at position 261 of SEQ ID NOs: 7 and 8 is believed to contribute, together with the modified HVR1 residues, to nuclease specificity. 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.

[0310] 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 the HVR2 region, except at 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 contains modifications relative to the wild-type I-CreI sequence (SEQ ID NO: 1) at positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 48, 50, 68, 70, 75, and 77. The TRC 1-2L.1592 meganuclease also contains modifications relative to wild-type I-CreI at positions 71, 72, and 73. It is also noteworthy 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, together with the modified HVR2 residues, to the specificity of the nuclease. The HVR2 region of TRC1-2L.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.

[0311] 2. Optimization of First-Generation TRC1-2 Nucleases

[0312] The recognition site specificity of the previously reported TRC 1-2x.87EE meganuclease was evaluated using a method very similar to GUIDE-seq (Tsai et al. (2015), Nat Biotechnology 33:187-197), but it was adjusted to discover potential off-target sites of meganucleases. Generally, potential off-target sites are identified by capturing probe oligonucleotides in double-strand DNA breaks. The TRC 1-2 meganuclease generates 3'-overhangs of 4 base pairs, so the probe oligonucleotides also contain randomized 4-base pair overhangs to improve ligation efficiency at sites more likely to be generated by nuclease cleavage.

[0313] Specificity analysis of TRC 1-2x.87EE identified multiple potential off-target sites in human T cells. These off-targets could be divided into two related categories: unique targets that were hit at high frequency and repetitive targets that were hit at low frequency. The key amino acids involved in the recognition of these off-targets were re-randomized. Subsequently, co-selection of the expected cleavage site and counter-selection of the off-target sites without cleavage were performed. The off-target sites were alternated between consecutive selection rounds to isolate and distinguish the two off-target answers (i.e., nucleases). The two off-targets used were Off1: 5’-TGGCCTGGAGaAACAgtgtaaa-3’ (SEQ ID NO:16), which was a low-frequency cleavage but highly repetitive site in the genome; and Off2: 5’-cGGCCTGtAGtAcaggAcCTGA-3’ (SEQ ID NO:17), which was a frequently hit, unique off-target (lowercase letters indicate mismatches with the expected site). Multiple nuclease libraries were used.

[0314] After selection, 96-well plates of isolated clones from each successful library were prepared to isolate plasmid DNA. Each plasmid DNA was individually transfected into CHO cells containing an integrated target site interrupted between two direct repeats in the GFP gene. Cleavage of the target site led to repair of the GFP gene by single-strand annealing, and the frequency of cleavage of the target site could be counted by counting the number of GFP-positive cells on a flow cytometer. We analyzed the nuclease plasmids against cells with the expected site and the Off1 target site. In this way, we could evaluate which nucleases still cleaved the expected site but best discriminated against the off-targets. We identified five candidates. Three candidates were re-isolated from the original library of TRC 1-2: L.1462, L.1466, and L.1469. All three answers were unique but related to each other. Two candidates were isolated from TRC library 2: L.1108 and L.1118. Each of these candidates represents an intermediate nuclease in the development of the second-generation nucleases of the present invention.

[0315] 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 randomization by PCR was introduced into both. The new libraries employed a similar selection strategy; selection was performed simultaneously for the intended site and against Off1 or Off2. Off-targets were alternated between selection rounds. Single answers were generated from the selection in 96-well plates and tested in the CHO iGFFP assay to determine cleavage at both the intended site and Off1 and Off2. Several new nucleases were identified from this additional optimization round. 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 the new nucleases showed strong activity against the intended target and strong discrimination against the two off-targets (described below). The new nucleases were subjected to an oligonucleotide capture assay (described further below) to determine potential off-target sites and demonstrated a reduced number of potential off-targets overall, and in particular, L.1592 had few potential reasonable off-target sites. L.1108, L.1469, L.1592, L.1775, and L.1843 were further evaluated for 7 days in the iGFFP assay to determine the stability of the GFP signal over time, which is a general measure of toxicity. L.1469, L.1592, L.1775, and L.1843 were further tested for function in primary T cells.

[0316] 3. Evaluation of TRC 1-2 Recognition Sequence Cleavage and Off-Target Cleavage

[0317] 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 ). For this analysis, a pair of CHO cell reporter lines was prepared that carried a non-functional green fluorescent protein (GFP) gene expression cassette integrated into the cell genome. The GFP gene in each cell line was interrupted by a pair of recognition sequences such that intracellular cleavage of either recognition sequence by the meganuclease would stimulate a homologous recombination event, resulting in the production of a functional GFP gene. In both cell lines, one of the recognition sequences was derived from the TRC 1-2 gene and the second recognition sequence was specifically recognized by a control meganuclease called "CHO 23 / 24". The 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".

[0318] Transfect TRC 1-2 cells with a plasmid DNA encoding one of the engineered meganucleases TRC 1-2 (e.g., TRC 1-2x.87EE, TRC 1-2L.1592, TRC1-2L.1775 or TRC 1-2L.1843) or the CHO 23 / 34 meganuclease. Transfect 4e5 CHO cells with 50 ng plasmid DNA in a 96-well plate using Lipofectamine 2000 (ThermoFisher) according to the manufacturer's instructions. Forty-eight hours after transfection, assess the cells by flow cytometry to determine the percentage of GFP-positive cells compared to untransfected negative control (1-2bs). All TRC 1-2 meganucleases were found to generate GFP-positive cells in the cell line containing the TRC 1-2 recognition sequence at frequencies significantly exceeding the negative control and comparable to or exceeding that of the CHO 23 / 24 positive control, indicating that each TRC 1-2 meganuclease can effectively recognize and cleave the expected TRC 1-2 recognition sequence in the cells ( Figure 4 A-4C).

[0319] Alternatively, the TRC 1-2 meganucleases were also transfected into TRC Off1 and TRC Off2 cells, which contain reverse-selected off-target sequences between GFP direct repeats. Different from the expected target site TRC 1-2 CHO cells, the meganucleases required in TRCOff1 and TRC Off2 CHO cells only have background levels of GFP-positive cells because it can distinguish between cleavage of off-target sequences. In these experiments, the CHO 23-24 target site served as a positive control, indicating that GFP can still be generated if the CHO23-24 meganuclease cleaves the target site. Compared to TRC 1-2x.87EE, the new meganucleases showed a significant improvement (i.e., increase) in discrimination against Off1 and Off2 target sites, with the %GFP levels comparable to the TRC 1-2bs negative control ( Figure 5 A-5C).

[0320] The efficacy of the engineered meganucleases TRC 1-2.L1469, L.1592, L.1775 and L.1843 was also determined in a time-dependent manner 2, 5 and 7 days after introducing the meganuclease mRNA into TRC 1-2 cells. In this study, TRC 1-2 cells (1.0x10 6 ) were used with 1x10 6Copy of the meganuclease mRNA electroporation. At 48 hours post transfection, cells were evaluated 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 a comparable percentage of GFP positive relative to CHO23-24 ( Figure 6 and Figure 7 ). Only L.1469 showed a trend of decreasing GFP positive cells over time, indicating that it had some unresolved toxicity issues, which were improved in subsequent optimizations. The remaining nucleases showed stable or increasing GFP positive cells over time, at levels equal to or higher than the CHO 23-24 control.

[0321] The extended iGFFP assay was also used to evaluate discrimination of the same set of meganucleases against two off-targets, Off1 and Off2, over a 7-day period. In this case, cells containing Off1 or Off2, as well as CHO 23-24, were electroporated with 1x10 6 copies of meganuclease mRNA per cell using a BioRad Gene Pulser Xcell according to the manufacturer's instructions. At 2, 5, and 7 days post transfection, cells were evaluated 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 nuclease showed improved discrimination against off-targets compared to TRC 1-2x.87EE ( Figure 8 A and 8B). L.1592 demonstrated minimal cleavage of Off1 or Off2, comparable to mock control cells. L.1469 showed some detectable cleavage of Off1 and Off2, but it was far lower than that observed with TRC 1-2x.87EE. L.1775 and L.1843 showed improvement in discrimination against off-targets compared to their parental L.1469.

[0322] 4. Oligonucleotide Capture Assays and Off-Target Cleavage Analysis

[0323] In these studies, oligonucleotide capture assays were used to identify off-target cleavage induced by the TRC 1-2 meganuclease. Similar to GUIDE-seq, the oligonucleotide capture assay identifies potential off-target sites generated by the TRC 1-2 meganuclease by capturing oligonucleotides at the break sites within cellular genomic DNA. GUIDE-seq was developed for DNA breaks generated by CRISPR-Cas9, and there were some key modifications to the chemistry and analysis to apply the technique to the current nuclease. Different from CRISPR-Cas9, the engineered meganucleases of the present invention generate 3'-overhangs of four base pairs. To accommodate this difference, the oligonucleotides used in the oligonucleotide capture have random four-base pair overhangs that can be compatible with the overhangs generated by the TRC 1-2 meganuclease. Higher insertion frequencies were observed due to the higher efficiency of ligating sticky ends rather than blunt ends. 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 adapters were ligated to the sheared DNA, and PCR was used to amplify any DNA fragments containing an adapter at one end and a captured oligonucleotide at the other end. The amplified DNA was purified, and a sequencing library was prepared using a standard commercial kit.

[0324] The sequencing library was run on an Illumina MiSeq using the V2 2x150 kit. The data were filtered and the valid sites of the captured oligonucleotides were analyzed, and potential off-target sites were predicted. Similarly, the experimental protocol needed to adjust the PAM search from that used for CRISPR-Cas9 to the TRC 1-2 meganuclease search. The developed software checks each sequence to ensure that there are adapters and captured oligonucleotides 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 scanned for grouped sequences within windows of thousands of base pairs to search for potential TRC1-2 meganuclease sites.

[0325] 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 allowed gaps. The middle four-base pair is not considered in off-target selection because TRC 1-2 meganucleases can generally tolerate higher degeneracy at these positions in the target site. The software outputs a list of potential off-target sites and the number of base mismatches in the combined half-sites, but does not count mismatches in the middle four-base pair. Unlike CRISPR-Cas9 which eliminates any off-targets identified with more than six mismatched base pairs, this software does not eliminate any off-targets based on an arbitrary mismatch filter. Instead, background noise generated from random capture of oligonucleotides at vulnerable points or hotspots within the genome can be reduced in two ways. First, untreated mock samples can also be run by oligonucleotide capture, and windows of integration sites without nuclease can be subtracted from samples containing nuclease. We also found that running the assay in triplicate and eliminating any sites that do not replicate in at least two of the three replicates is a good way to empirically eliminate random integration noise.

[0326] Although read fragment counts are not directly related to the cleavage frequency at a particular position, they can generally highlight off-targets that may be of more concern or more efficient because they occur more frequently. Figure 9 A way to graphically visualize oligonucleotide capture data as a measure of the number of potentially valid off-target sites is shown. Each off-target generated by a particular nuclease is plotted according to the number of unique sequence reads of the probe oligonucleotide captured at that site. The expected sites should have the highest read counts, which is the case for all tested TRC 1-2 meganucleases. Better nucleases remove sites with higher counts and have fewer points above the background noise on the far left of the graph. Using this graph, it can be clearly seen, for example, that TRC 1-2L.1592 removes more sites with higher read counts than the first-generation TRC 1-2x.87EE.

[0327] Other visualization methods enable us to view oligonucleotide capture data not only by the number of reads recovered at a particular site, but also by the number of mismatches between the putative off-target site and the expected site. This allows for a more accurate determination of true oligonucleotide integration sites compared to random integration or sequencing noise. In Figure 10In it, off-target sites are plotted according to the number of read segments they align with on the X-axis, 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. The boxes represent the regions with the highest confidence. Off-targets within these boxes have high aligned read segment counts or very high similarity to the expected site, either of which reduces the likelihood that the site is background noise. Comparing the sites in the confidence regions, Figure 10 demonstrates that the specificity of the optimized meganucleases, especially TRC 1-2L.1592, is improved compared to TRC 1-2x.87EE. TRC 1-2L.1592 shows a decrease in the number of sites with high read segment counts and a decrease in sites that are more similar to the expected ones.

[0328] Example 2

[0329] In Vitro Analysis of Optimized TRC 1-2 Megal nucleases

[0330] 1. Evaluation of Gene Editing Efficiency, Post-Editing Amplification, and Differentiation

[0331] In the first set of experiments, four optimized second-generation TRC 1-2 meganucleases were screened for their gene editing efficiency and post-editing amplification and differentiation potential. Three different operators each evaluated all nuclease variants in T cells obtained from different healthy human T cell donors. The apheresis materials were from donors K708, K799, and K6784 of Key Biologics (Memphis, TN). K708 and K6784 T cells were processed according to the following protocol: T cell enrichment using a human CD3 positive selection reagent (StemCell Technologies), stimulation using ImmunoCult anti-CD2 / CD3 / CD28 (StemCell Technologies), and nuclease RNA delivery using a 4D NucleoFEctor (Lonza). T cells from K799 were processed according to the following protocol: T cell enrichment using CD4 and CD8 microbeads and a CliniMACS cell separator (Miltenyi Biotec), stimulation using TransAct (Miltenyi), and nuclease RNA delivery using a MaxCyte-GT.

[0332] 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 the CAR gene to be inserted into the TRC 1-2 cleavage site. Control cultures not receiving AAV were assembled in parallel.

[0333] On days 4 and 8 post-editing, total culture cellularity was determined using a NucleoCounter NC-200 (ChemoMetec). Editing efficiency was determined by staining culture samples with an antibody against human CD3-PE (BioLegend clone UCHT1) and anti-FMC63 scFv-AlexaFluor647 (a new clone produced in-house and conjugated). Differentiation was assessed by comparing the frequencies 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).

[0334] The results of these experiments are summarized in Figure 11 The knockout frequencies of the endogenous T cell receptor for each nuclease in 3 different donors were determined (measured by T cells transitioning from a CD3-positive to a CD3-negative phenotype). For all 3 donors tested (and using two cell preparation methods), both TRC 1-2L.1592 and L.1775 produced knockout cells with similar or higher efficiency compared to 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. Increased editing of the TRC 1-2 recognition sequence was associated with an increased insertion rate of the CAR gene. In all three donors, L.1592 and L.1775 supported equivalent or superior editing and insertion frequencies.

[0335] On day 8 post-editing, cell count data were 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 after 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 by donor.

[0336] CD4:CD8 ratio and memory subset data were also captured on day 8 post-editing. No major perturbation of the CD4:CD8 ratio was 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, when edited with L.1592, L.1775, or L.1843, an equal or higher frequency of cells maintained the central memory phenotype.

[0337] These studies indicate that three of the four optimized nucleases are superior to TRC 1-2x.87EE in terms of editing efficiency, cell expansion, and differentiation characteristics. One nuclease (L.1469) performed less well than x.87EE. Among 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-editing expansion and accelerated the differentiation of T cells in culture. Variant L.1843 allowed the greatest amount of post-editing expansion and maintained a favorable central memory frequency but was less efficient than L.1775 or L.1592 in terms of knockout frequency. Unexpectedly, using all three criteria, L.1592 represents an improvement over the first-generation x.87EE.

[0338] 2. Analysis of Oligonucleotide Capture Assays and Off-Target Cleavage

[0339] Using the method described in Example 1 above, oligonucleotide capture was performed on three replicates of T cells obtained from each of three donors. The results of 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 on-target site. Putative sites with more than 7 mismatches to the expected target were removed because sites with more than 7 mismatches have not been shown to be cut by TRC 1-2L.1592 in previous studies. The expected on-target sites for each sample are highlighted with circles. The number of mismatches compared to the expected target is represented by the darkness of each circle, with fewer mismatches having a darker color. This figure represents oligonucleotide capture data where mock background has not been removed and the read counts are normalized to the number of unique reads for each sample to account for differences in the total number of reads recovered. As shown, TRC 1-2L.1592 shows a small number of sites with higher read counts, as well as a small number of sites that are more similar to the expected sites when used for editing and targeted insertion in the CART cell population.

[0340] 3. In Vitro Studies of Editing Efficiency, Amplification, and Cytokine Secretion

[0341] In a second set of in vitro studies, the efficiency of the second-generation optimized TRC 1-2 meganuclease in editing T cells, the ability of the edited T cells to expand after editing, and the ability of the CAR T cells generated with the nuclease variant to respond to encounter with antigen-bearing target cells were evaluated.

[0342] Apheresis materials were derived from donor K708 of Key Biologics (Memphis, TN), and T cells were enriched using a human CD3 positive selection reagent (StemCell Technologies), stimulated with ImmunoCult anti-CD2 / CD3 / CD28 (StemCell Technologies), and nuclease RNA was delivered using a 4D NucleoFector (Lonza). Samples were run in triplicate in parallel.

[0343] The editing efficiency, expansion, and differentiation of three optimized meganucleases (TRC 1-2L.1592, L.1775, and L.1843) were compared with the progenitor meganuclease TRC 1-2x.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 meganucleases, and then immediately transduced with an AAV6 vector encoding the CAR gene to be inserted into the TRC 1-2 cleavage site. On days 4 and 8 post-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 evaluated using anti-CD3-PE (BioLegend clone UCHT1), and CAR knock-in was measured using anti-FMC62 scFv-AlexaFluor647 (a new clone produced in-house and conjugated).

[0344] Proliferation, cytotoxicity, and cytokine production were evaluated by co-culturing CAR T cells with the 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 controls. 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 evaluated by staining cultured cell samples with anti-CD4-APC (BioLegend clone OKT4), anti-CD8-FITC (BioLegend clone RPA-T8), and anti-CD19-PE (BioLegend clone HIB19), and acquiring fluorescence data and cell counts using a CytoFLEX-LX.

[0345] Compared to T cells without RNA electroporation (mock control), T cells edited with the TRC 1-2 meganucleases x.87EE and L.1775 had a ~50% reduction in total culture cell viability on day 8 ( Figure 13 A). Cultures edited with L.1592 or L.1843 did not show such a large reduction in total culture cell viability. When considering editing efficiency and calculating the total number of edited cells generated in this 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 fewest. This pattern was also observed when measuring the number of CAR+ / TCR- cells in the cultures ( Figure 13 C).

[0346] When CAR T cells were co-cultured with antigen-bearing target cells, CAR T cells generated with TRC 1-2x.87EE expanded nearly three-fold in the input number (– defined by the horizontal dotted line Figure 14 ). Unexpectedly, CAR T cells generated with the optimized nuclease 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, the proliferation of x.87EE- and L.1843-edited CAR T cells decreased by approximately 1 / 2 relative to the 1:1 ratio. This was not observed in CAR T cells generated with L.1775 or L.1592, which were found to have significantly better performance than other TRC 1-2 nucleases (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 generated with the optimized nuclease eliminated Raji cells significantly better than cells generated with x.87EE.

[0347] Analysis of the co-culture supernatants showed that higher levels of effector cytokines were produced when CAR T cells were prepared with the optimized nuclease rather than x.87EE. L.1592-edited CAR T cells secreted the highest levels of IL-2, TNFα, IFNγ, and granzyme B ( Figure 16 A-16D), as well as the second highest level of perforin ( Figure 16 E). In the case of IL-2 and TNFα, the difference in cytokine production between x.87EE-edited and L.1592-edited CAR T cells was 2-3-fold, while all other differences were small.

[0348] Overall, the optimized TRC 1-2 meganucleases L.1775, L.1592, and L.1843 were functionally superior to x.87EE. This was true in terms of the relative ability of the nucleases to support CAR T cell manufacturing ( Figure 13 ) and the ability of the CAR T cells to respond to encounter with their target antigen ( Figures 14 - 16 ). It could be concluded from multiple experiments that while L.1775 generally supported the highest editing efficiency (knockout frequency), and L.1843 allowed the greatest expansion of T cells after editing, L.1592 combined the second highest editing efficiency with the highest or second highest expansion to yield the highest overall number of CAR T cells. Importantly, CAR T cells generated with L.1592 showed functional advantages (proliferation, target cell killing, and cytokine production) compared to other optimized meganucleases.

[0349] 4. In Vitro Residence Time of Optimized TRC 1-2 Megal nucleases

[0350] Further studies were conducted to determine whether the residence time of the optimized second-generation TRC 1-2 meganuclease in vitro is shorter than that of the first-generation TRC 1-2x.87EE. A shorter residence time may be advantageous in the context of potential reduction of gene editing and off-target cleavage.

[0351] In these studies, T cells were obtained from apheresis blood separation products (Key Biologics) by magnetic enrichment of CD4+ and CD8+ cells using CD4 and CD8 microbeads and an LD column (Miltenyi). The cells were activated for three days with anti-CD3 / anti-CD28 TransAct reagent (Miltenyi) in Xuri medium (GE) containing 5% FBS (GE Hyclone), 10 ng / ml IL-2 (Cellgenix), and 1% antibiotic / antifungal solution (Gibco). Then, using the MaxCyte electroporation system, the cells were electroporated with in vitro transcribed mRNA (Trilink) encoding either the TRC 1-2x.87EE or TRC 1-2L.1592 meganuclease, 1 μg mRNA per 1e6 cells. Subsequently, in serum-free Xuri medium containing 30 ng / ml IL and 1% antibiotic / antifungal solution, the cells were transduced by homologous recombination (SAB Tech) with a recombinant AAV6 vector carrying a donor template encoding an anti-CD19 chimeric antigen receptor designed for insertion at the TRC 1-2 locus. Six hours after electroporation, the samples were quantified and resuspended in Xuri medium containing 5% FBS, 30 ng / ml IL-2, and 1% antibiotic / antifungal solution. At the 96-hour time point, residual unedited CD3+ T cells were removed from the TRC electroporated group by magnetic depletion using an LD column, CliniMACS buffer, and CD3 microbeads (Miltenyi). Then, for the remaining experiments, the cells were cultured at 37 °C in Xuri medium + 5% FBS / 1% anti-anti + 10 ng / ml IL-15 and IL-21.

[0352] At 6 hours, 24 hours, 48 hours, 96 hours, and 168 hours after electroporation, T cell samples were quantified and equal amounts of live cells were pelleted and resuspended in RIPA buffer (EMD Millipore) supplemented with protease inhibitor (Roche), mixed well, and either frozen or incubated on ice for 30 minutes before further processing as described below for Western blotting.

[0353] Activate mock cells from the same donor and culture them in the same medium as the nuclease-treated group, and harvest them 24 hours after electroporation of the nuclease-treated group.

[0354] For Western blot analysis, centrifuge the lysates and transfer the supernatants to new tubes and place on ice. Determine protein concentration by BCA assay (Pierce), and 15 μg of total protein per sample is for sample buffer + DTT (NuPage), incubated at 90 °C for 10 minutes. Load 5 μg of each sample into each well of the gel. A single mock sample from the 24-hour time point after electroporation is used as a control. After electrophoresis, transfer the samples (NuPage electrophoresis system and reagents) to a PVDF membrane (Novex). Block the membrane with 5% non-fat dry milk in TBS-T and stain with primary antibodies:

[0355] Blot primary antibody

[0356] A Rabbit polyclonal anti-nuclease (proprietary to Precision BioSciences, used at 1:6500)

[0357] B Mouse anti-β-actin (Sigma, used at 1:15000)

[0358] Wash the membrane 6 times and then incubate with the appropriate secondary antibodies:

[0359] Blot secondary antibody

[0360] A Goat anti-rabbit HRP (Invitrogen, used at 1:50000)

[0361] B Goat anti-mouse HRP (Invitrogen, used at 1:75000)

[0362] After the wash step, expose the membrane to ECL Prime (Amersham), wrap in Saran wrap, and capture images using a UVP ChemiDoc-It 815 imager.

[0363] As Figure 17As shown, as expected, nuclease expression was not detected in the mock samples. In samples electroporated with mRNA encoding the TRC1-2x.87EE or TRC1-2L.1592 nucleases, nuclease proteins were highly expressed at the earliest time point analyzed, 6 hours post-electroporation. At 24 hours post-electroporation, the proteins were still detectable; however, expression of both nucleases was observed to be significantly lower than at 6 hours post-electroporation, with TRC 1-2L.1592 being significantly lower than TRC 1-2x.87EE at that time point. In samples treated with TRC 1-2L.1592 mRNA, nuclease proteins were undetectable at 48 hours post-electroporation or at subsequent time points, while TRC 1-2x.87EE protein expression was still detectable at that time point. Actin expression was consistent across all samples and time points, indicating equal amounts of protein were added to each sample.

[0364] These studies showed that the TRC1-2x.87EE and TRC1-2L.1592 nucleases were expressed at high levels 6 hours post-mRNA electroporation. However, expression of TRC 1-2L.1592 in T cells decreased more rapidly than that of TRC 1-2x.87EE. As Figure 11 shown, compared to TRC 1-2x.87EE, TRC 1-2L.1592 did not show reduced gene editing efficiency despite its shorter expression duration. Retaining high gene editing activity while reducing expression duration is a desirable property of TRC 1-2L.1592 and represents an unexpected and favorable improvement compared to TRC 1-2x.87EE, as these properties are associated with enhanced (i.e., increased) tolerance, higher T cell proliferation capacity, and lower off-target activity of TRC 1-2L.1592 compared to TRC1-2x.87EE.

[0365] Example 3

[0366] Evaluation of Optimized TRC1-2 Megal nucleases in CART Generation

[0367] The TRC 1-2L.1592 meganuclease was further evaluated in large-scale processes to determine whether the scale-up production of CAR T cells was improved relative to the first-generation TRC 1-2x.87EE meganuclease.

[0368] The large-scale process for generating allogeneic CAR T cells using TRC 1-2x.87EE starts with a fresh Leukopak from a healthy pre-screened donor. The Leukopak product is washed to remove platelets before immunomagnetic enrichment of the target T cells. The enriched T cells are then washed into a growth medium and activated using an activation reagent. After a 3-day activation period, the cells are washed and concentrated in an 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 a growth medium containing an AAV vector encoding the CAR insert gene. After the expansion period, the cells are collected on day 8 and immunomagnetic depletion of the CD3-positive population is performed. After depletion, the target CD3-negative cells are re-expanded in the growth medium for some time. 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 using TRC1-2L.1592 is performed substantially the same as that described for TRC 1-2x.87EE, except that the growth medium formulation in the TRC 1-2L.1592 trial is animal origin-free (AOF).

[0369] The total number of live cells is determined ([[]] Figure 18 ) at key time points during the production process. From day 0 to the depletion step on day 8, the cell numbers are comparable. However, due to the significantly higher T cell receptor knockout 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 during the TRC 1-2x.87EE process. The expansion rates between days 8 and 13 are similar, resulting in a total of approximately twice as many viable cells on day 13.

[0370] The CD3 knockout efficiency (i.e., an indicator of endogenous T cell receptor knockout) is determined by flow cytometry on day 8 of each production process ([[[]] Figure 19 ). Unexpectedly, the percentage of CD3-negative, gene-edited cells (among total live cells) is nearly 20% higher in the TRC 1-2L.1592 process than in the TRC 1-2x.87EE process.

[0371] Finally, the CAR knock-in efficiency is measured by flow cytometry at three key time points during each production process ([[[]] Figure 20)。Unexpectedly, the percentage of CAR-positive transduced cells in (CD3-negative cells) during the TRC 1-2L.1592 process was approximately 25% higher compared to the TRC 1-2x.87EE process. Between the end of the 8th day and the 13th day, the percentage of CAR knock-in was stable for both processes, resulting in a similarly higher percentage of CAR-positive cells at the end of the TRC 1-2L.1592 process.

[0372] In summary, these studies surprisingly showed that the TRC 1-2L.1592 nuclease significantly increased the quantity as well as the quality of the final allogeneic cell therapy product. TRC 1-2L.1592 more effectively knocked 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 approximately two-fold. Additionally, TRC1-2L.1592 potentially provided an improved environment for homologous recombination of the CAR gene insert at the targeted double-strand break, as demonstrated by the improved CAR knock-in efficiency. The increase in the CAR-positive percentage led to a significantly higher drug product purity, with fewer CAR-negative cells.

Claims

1. An engineered meganuclease that recognizes and cleaves a recognition sequence comprising SEQ ID NO:5 within the human T cell receptor (TCR) α constant region gene, wherein the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, wherein the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region, and wherein the HVR2: (a) has at least 81% sequence identity with the amino acid sequence corresponding to residues 24-79 of SEQ ID NO:7; or (b) has at least 86% sequence identity with the amino acid sequence corresponding to residues 24-79 of SEQ ID NO:

8.

2. The engineered meganuclease according to claim 1, wherein 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.

3. The engineered meganuclease according to claim 1 or 2, wherein the HVR2 region comprises residues corresponding to residues 48, 50, 71, 72 and 73 of SEQ ID NO:

7.

4. The engineered meganuclease according to any one of claims 1-3, wherein the HVR2 region comprises residues corresponding to residues 48 and 50 of SEQ ID NO:

8.

5. The engineered meganuclease according to any one of claims 1-4, wherein 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 8.

6. The engineered meganuclease according to any one of claims 1-5, wherein the HVR2 region comprises Y, R, K or D at the residue corresponding to residue 66 of SEQ ID NO:7 or 8.

7. The engineered meganuclease according to any one of claims 1-6, wherein the HVR2 region comprises residues 24-79 of SEQ ID NO:7 or 8.

8. The engineered meganuclease according to any one of claims 1-7, wherein the second subunit comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence corresponding to residues 7-153 of SEQ ID NO:7 or 8.

9. The engineered meganuclease according to any one of claims 1 to 8, wherein the second subunit comprises G, S or A at the residue corresponding to residue 19 of SEQ ID NO:7 or 8.

10. The engineered meganuclease according to any one of claims 1-9, wherein the second subunit comprises E, Q or K at a residue corresponding to residue 80 of SEQ ID NO: 7 or 8.

11. The engineered meganuclease according to any one of claims 1-10, wherein the second subunit comprises an amino acid sequence having at least 80% sequence identity with residues 7-153 of SEQ ID NO: 7 or 8.

12. The engineered meganuclease according to any one of claims 1-11, wherein the first subunit comprises an amino acid sequence having at least 80% sequence identity with residues 198-344 of SEQ ID NO: 7 or 8, and wherein the second subunit comprises an amino acid sequence having at least 80% sequence identity with residues 7-153 of SEQ ID NO: 7 or 8.

13. The engineered meganuclease according to any one of claims 1-12, wherein the second subunit comprises G, S or A at a residue corresponding to residue 19 of SEQ ID NO: 7 or 8.

14. The engineered meganuclease according to any one of claims 1-13, wherein the second subunit comprises E, Q or K at a residue corresponding to residue 80 of SEQ ID NO: 7 or 8.

15. The engineered meganuclease according to any one of claims 1-14, wherein the second subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 7 or 8.

16. The engineered meganuclease according to any one of claims 1-15, wherein the second subunit comprises a residue corresponding to residue 139 of SEQ ID NO: 7 or 8.

17. The engineered meganuclease according to any one of claims 1-16, wherein the second subunit comprises residues 7-153 of SEQ ID NO: 7 or 8.

18. The engineered meganuclease according to any one of claims 1-17, wherein the HVR1 region comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7 or 8.

19. The engineered meganuclease according to any one of claims 1-18, wherein 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.

20. The engineered meganuclease according to any one of claims 1-19, wherein the HVR1 region comprises Y, R, K or D at a residue corresponding to residue 257 of SEQ ID NO: 7 or 8.

21. An engineered meganuclease according to any one of claims 1-20, wherein the HVR1 region comprises residues 215-270 of SEQ ID NO: 7 or 8.

22. An engineered meganuclease according to any one of claims 1-21, wherein the first subunit comprises an amino acid sequence having at least 80% sequence identity with residues 198-344 of SEQ ID NO: 7 or 8.

23. An engineered meganuclease according to any one of claims 1-22, wherein the first subunit comprises G, S or A at the residue corresponding to residue 210 of SEQ ID NO: 7 or 8.

24. An engineered meganuclease according to any one of claims 1-23, wherein the first subunit comprises E, Q or K at the residue corresponding to residue 271 of SEQ ID NO: 7 or 8.

25. An engineered meganuclease according to any one of claims 1-24, wherein the first subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 7 or 8.

26. An engineered meganuclease according to any one of claims 1-25, wherein the first subunit comprises residues 198-344 of SEQ ID NO: 7 or 8.

27. An engineered meganuclease according to any one of claims 1-26, wherein the engineered meganuclease comprises a linker, wherein the linker covalently joins the first subunit and the second subunit.

28. An engineered meganuclease according to any one of claims 1-27, wherein the engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 7 or 8.

29. An engineered meganuclease according to any one of claims 1-28, wherein compared to the TRC 1-2x.87EE meganuclease as shown in SEQ ID NO: 9, the engineered meganuclease exhibits at least one of the following optimized characteristics: improved specificity, reduced intracellular duration, and increased modification efficiency of the human TCRα constant region gene.

30. A polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease according to any one of claims 1-29.

31. The polynucleotide according to claim 30, wherein the polynucleotide is mRNA.

32. The polynucleotide according to claim 31, wherein the mRNA is a polycistronic mRNA encoding an engineered meganuclease according to any one of claims 1-29 and at least one other polypeptide or nucleic acid.

33. A recombinant DNA construct comprising the polynucleotide according to claim 30.

34. The recombinant DNA construct according to claim 33, wherein the recombinant DNA construct encodes a viral vector.

35. The recombinant DNA construct according to claim 34, wherein the viral vector is an adenovirus vector, a lentivirus vector, a retrovirus vector, or an adeno-associated virus (AAV) vector.

36. The recombinant DNA construct according to claim 34 or claim 35, wherein the viral vector is a recombinant AAV vector.

37. A viral vector comprising the polynucleotide according to claim 30.

38. The viral vector according to claim 37, wherein the viral vector is an adenovirus vector, a lentivirus vector, a retrovirus vector, or an AAV vector.

39. The viral vector according to claim 37 or claim 38, wherein the viral vector is a recombinant AAV vector.

40. A method of generating a genetically modified eukaryotic cell comprising inserting an exogenous target sequence into the chromosome of the eukaryotic cell, the method comprising introducing one or more nucleic acids into the eukaryotic cell, the nucleic acids comprising: (a) a first nucleic acid encoding an engineered meganuclease according to any one of claims 1-29, wherein the engineered meganuclease is expressed in the eukaryotic cell; and (b) a second nucleic acid comprising the target sequence; wherein the engineered meganuclease generates a cleavage site in the chromosome at the recognition sequence comprising SEQ ID NO: 5; and wherein the target sequence is inserted into the chromosome at the cleavage site.

41. The method according to claim 40, wherein the second nucleic acid further comprises a sequence homologous to the sequence flanking the cleavage site, and the target sequence is inserted at the cleavage site by homologous recombination.

42. The method according to claim 40 or claim 41, wherein the cell surface expression of the endogenous T cell receptor is reduced on the genetically modified cell as compared to an unmodified control cell.

43. The method according to any one of claims 40-42, wherein the eukaryotic cell is a human T cell or a cell derived therefrom, or a human NK cell or a cell derived therefrom.

44. The method according to any one of claims 40-43, wherein the target sequence comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor.

45. The method according to claim 44, wherein the chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand-binding domain specific for a tumor-specific antigen.

46. The method according to any one of claims 40-45, wherein at least the first nucleic acid is introduced into the eukaryotic cell by mRNA.

47. The method according to any one of claims 40-46, wherein at least the second nucleic acid is introduced into the eukaryotic cell by a viral vector.

48. The method according to claim 47, wherein the viral vector is an adenovirus vector, a lentivirus vector, a retrovirus vector, or an AAV vector.

49. The method according to claim 47 or claim 48, wherein the viral vector is a recombinant AAV vector.

50. A method for generating a genetically modified eukaryotic cell, which comprises inserting an exogenous target sequence into the chromosome of the eukaryotic cell, the method comprising: (a) introducing an engineered meganuclease according to any one of claims 1-29 into the eukaryotic cell; and (b) introducing a nucleic acid comprising the target sequence into the eukaryotic cell; wherein the engineered meganuclease generates a cleavage site in the chromosome at the recognition sequence comprising SEQ ID NO: 5; and wherein the target sequence is inserted into the chromosome at the cleavage site.

51. The method according to claim 50, wherein the nucleic acid further comprises a sequence homologous to the sequence flanking the cleavage site, and the target sequence is inserted at the cleavage site by homologous recombination.

52. The method according to claim 50 or claim 51, wherein the cell surface expression of the endogenous T cell receptor is reduced on the genetically modified cell when compared to an unmodified control cell.

53. The method according to any one of claims 50-52, wherein the eukaryotic cell is a human T cell or a cell derived therefrom, or a human NK cell or a cell derived therefrom.

54. The method according to any one of claims 50-53, wherein the target sequence comprises a coding sequence of a chimeric antigen receptor or an exogenous T cell receptor.

55. The method according to claim 54, wherein the chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand-binding domain specific for a tumor-specific antigen.

56. The method according to any one of claims 50-55, wherein the nucleic acid is introduced into the eukaryotic cell by a viral vector.

57. The method according to claim 56, wherein the viral vector is an adenovirus vector, a lentivirus vector, a retrovirus vector or an AAV vector.

58. The method according to claim 55 or claim 56, wherein the viral vector is a recombinant AAV vector.

59. A method for generating a genetically modified eukaryotic cell by disrupting a target sequence in the chromosome of a eukaryotic cell, the method comprising: introducing a nucleic acid encoding an engineered meganuclease according to any one of claims 1-29 into the eukaryotic cell, wherein the engineered meganuclease is expressed in the eukaryotic cell; wherein the engineered meganuclease generates a cleavage site in the chromosome at the recognition sequence comprising SEQ ID NO: 5, and wherein the target sequence is disrupted by non-homologous end joining at the cleavage site.

60. The method according to claim 59, wherein the cell surface expression of the endogenous T cell receptor is reduced on the genetically modified cell when compared to an unmodified control cell.

61. The method according to claim 59 or claim 60, wherein the eukaryotic cell is a human T cell or a cell derived therefrom, or a human NK cell or a cell derived therefrom.

62. The method according to any one of claims 59 - 61, wherein the nucleic acid is introduced into the eukaryotic cell via mRNA.

63. A method for generating a genetically modified eukaryotic cell by disrupting a target sequence in a eukaryotic cell chromosome, the method comprising: introducing an engineered meganuclease according to any one of claims 1 - 29 into a eukaryotic cell; wherein the engineered meganuclease generates a cleavage site in the chromosome at the recognition sequence comprising SEQ ID NO: 5, and wherein the target sequence is disrupted by non - homologous end joining at the cleavage site.

64. The method according to claim 63, wherein the cell surface expression of the endogenous T - cell receptor is reduced on the genetically modified cell when compared to an unmodified control cell.

65. The method according to claim 63 or claim 64, wherein the eukaryotic cell is a human T - cell or a cell derived therefrom, or a human NK - cell or a cell derived therefrom.

66. A genetically modified eukaryotic cell prepared by the method according to any one of claims 40 - 58.

67. The genetically modified eukaryotic cell according to claim 66, wherein the cell comprises a reduced off - target effect of the engineered meganuclease, a reduced duration of the engineered meganuclease in the eukaryotic cell, or both, compared to the TRC 1 - 2x.87EE meganuclease as shown in SEQ ID NO:

9.

68. A genetically modified eukaryotic cell prepared by the method according to any one of claims 59 - 65.

69. The genetically modified eukaryotic cell according to claim 68, wherein the cell comprises a reduced off - target effect of the engineered meganuclease, a reduced duration of the engineered meganuclease in the eukaryotic cell, or both, compared to the TRC 1 - 2x.87EE meganuclease as shown in SEQ ID NO:

9.

70. A population of genetically modified eukaryotic cells comprising a plurality of the genetically modified eukaryotic cells according to claim 66 or claim 67.

71. The population according to claim 70, 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% or up to 100% of the cells in the population are the genetically modified eukaryotic cells according to claim 66 or claim 67.

72. The population according to claim 70 or claim 71, wherein the genetically modified eukaryotic cell is a genetically modified human T - cell or a cell derived therefrom, or a genetically modified NK - cell or a cell derived therefrom.

73. The population according to any one of claims 70 - 72, wherein the target sequence comprises a coding sequence for a chimeric antigen receptor or an exogenous T - cell receptor.

74. The population according to claim 73, wherein the chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand-binding domain that is specific for a tumor-specific antigen.

75. The population according to any one of claims 70-74, wherein the cell surface expression of the endogenous T cell receptor on the genetically modified eukaryotic cell is reduced when compared to an unmodified control cell.

76. A population of genetically modified eukaryotic cells comprising a plurality of the genetically modified eukaryotic cells according to claim 68 or claim 69.

77. The population according to claim 76, 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% or up to 100% of the cells in the population are the genetically modified eukaryotic cells according to claim 68 or claim 69.

78. The population according to claim 76 or claim 77, wherein the genetically modified eukaryotic cell is a genetically modified human T cell or a cell derived therefrom, or a genetically modified NK cell or a cell derived therefrom.

79. The population according to any one of claims 76-78, wherein the genetically modified eukaryotic cell comprises a cell surface chimeric antigen receptor or an exogenous T cell receptor.

80. The population according to claim 79, wherein the chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand-binding domain that is specific for a tumor-specific antigen.

81. The population according to any one of claims 76-80, wherein the cell surface expression of the endogenous T cell receptor on the genetically modified eukaryotic cell is reduced when compared to an unmodified control cell.

82. A pharmaceutical composition 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 the genetically modified eukaryotic cell according to claim 66 or claim 67 or the population of genetically modified eukaryotic cells according to any one of claims 70-75.

83. The pharmaceutical composition according to claim 82, wherein the genetically modified eukaryotic cell or the population consists of a genetically modified human T cell or a cell derived therefrom, or a genetically modified NK cell or a cell derived therefrom.

84. The pharmaceutical composition according to claim 82 or claim 83, wherein the target sequence comprises a coding sequence of a chimeric antigen receptor or an exogenous T cell receptor.

85. The pharmaceutical composition according to claim 84, wherein the chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand-binding domain that is specific for a tumor-specific antigen.

86. The pharmaceutical composition according to any one of claims 82 - 85, wherein the cell surface expression of the endogenous T cell receptor on the genetically modified eukaryotic cell is reduced compared to an unmodified control cell.

87. A pharmaceutical composition 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 population of the genetically modified eukaryotic cells according to claim 68 or claim 69 or the genetically modified eukaryotic cells according to any one of claims 76 to 81.

88. The pharmaceutical composition according to claim 87, wherein the genetically modified eukaryotic cell is a genetically modified human T cell or a cell derived therefrom, or a genetically modified NK cell or a 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.

89. The pharmaceutical composition according to claim 87 or claim 88, wherein the genetically modified eukaryotic cell comprises a cell surface chimeric antigen receptor or an exogenous T cell receptor.

90. The pharmaceutical composition according to claim 89, wherein the chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand-binding domain specific for a tumor-specific antigen.

91. The pharmaceutical composition according to any one of claims 87 - 90, wherein the cell surface expression of the endogenous T cell receptor on the genetically modified eukaryotic cell is reduced when compared to an unmodified control cell.

92. A method for treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a population of the genetically modified eukaryotic cells according to any one of claims 66 - 69 or the genetically modified eukaryotic cells according to any one of claims 70 - 81.

93. The method according to claim 80, wherein the method comprises administering to the subject the pharmaceutical composition according to any one of claims 82 - 91.

94. The method according to claim 92 or claim 93, wherein the method is an immunotherapy for treating cancer in a subject in need thereof, and wherein the genetically modified eukaryotic cell is a genetically modified human T cell or a cell derived therefrom, or a genetically modified NK cell or a cell derived therefrom, and wherein the genetically modified eukaryotic cell comprises a cell surface chimeric antigen receptor or an exogenous T cell receptor, the cell surface chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand-binding domain specific for a tumor-specific antigen, and wherein the cell surface expression of the endogenous T cell receptor on the genetically modified eukaryotic cell is reduced compared to an unmodified control cell.

95. The method according to claim 94, wherein the cancer is selected from malignant epithelial cancer, lymphoma, sarcoma, blastoma, and leukemia.

96. The method according to claim 94 or claim 95, wherein the cancer is selected from cancers 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 lymphoma.

97. The method according to claim 96, wherein the cancer of B-cell origin is selected from B-cell lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin lymphoma, and multiple myeloma.

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