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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0646—Natural killers cells [NK], NKT cells
• • C—CHEMISTRY; METALLURGY
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
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  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/11—Exodeoxyribonucleases producing 5'-phosphomonoesters (3.1.11)
  • C12Y301/11002—Exodeoxyribonuclease III (3.1.11.2)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/50—Fusion polypeptide containing protease site
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
  • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
• • C—CHEMISTRY; METALLURGY
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  • C12N2510/00—Genetically modified cells

Definitions

• Cancer The global burden of cancer doubled between 1975 and 2000. Cancer is the second leading cause of morbidity and mortality worldwide, with approximately 14.1 million new cases and 8.2 million cancer related deaths in 2012.
• the most common cancers are breast cancer, lung and bronchus cancer, prostate cancer, colon and rectum cancer, bladder cancer, melanoma of the skin, non-Hodgkin lymphoma, thyroid cancer, kidney and renal pelvis cancer, endometrial cancer, leukemia, and pancreatic cancer.
• the number of new cancer cases is projected to rise to 22 million within the next two decades.
• the present disclosure contemplates, in part, a polypeptide comprising an I-Onul homing endonuclease (HE) variant that cleaves a target site in the human T cell receptor alpha (TCRa) gene, wherein the variant comprises the amino acid substitutions: L26I, R28D, N32R, K34N, S35E, V37N, G38R, S40R, E42S, G44R, V68K, A70T, G73S, N75R, S78M, K80R, L138M, T143N, S159P, S176A, C180H, F182G, I186K, S188V, S190G, K191T, L192A, G193K, Q195Y, Q197G, V199R, S201A, T203S, K207R, Y223S, K225R, S233R, D236E, and V238E of any one of SEQ ID NOs: 1-5, or a biological
• the polypeptide binds the polynucleotide sequence set forth in SEQ ID NO: 17.
• the zinc finger DNA binding domain comprises 2, 3, 4, 5, 6, 7, or 8 zinc finger motifs.
• the end-processing enzyme comprises Trex2 or a biologically active fragment thereof.
• the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 12, or a biologically active fragment thereof.
• a cell comprising a polypeptide contemplated herein is provided.
• the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
• SEQ ID NO: 6 is an amino acid sequence of an I-Onul LHE variant reprogrammed to bind and cleave a target site in exon 1 of the constant region of the human TCRa gene.
• SEQ ID NO: 15 is an mRNA sequence that encodes a megaTAL that binds and cleaves a target site in exon 1 of the constant region of the human TCRa gene.
• SEQ ID NO: 19 is an 11.5 RVD TALE DNA binding domain target site in exon 1 of the constant region of the human TCRa gene.
• SEQ ID NO: 20 is a megaTAL target site in exon 1 of the constant region of the human TCRa gene.
• modification of one or more TCRa alleles ablates or substantially ablates expression of the TCRa allele(s), decreases expression of the TCRa allele(s), and/or impairs, substantially impairs, or ablates one or more functions of the TCRa allele(s) or renders the TCRa allele(s) non-functional.
• TCRa functions include, but are not limited to, recruiting CD3 to the cell surface, MHC dependent recognition and binding of antigen, activation of TCRaP signaling.
• Genome editing compositions and methods contemplated in various embodiments comprise nuclease variants, designed to bind and cleave a target site in the human T cell receptor alpha (TCRa) gene.
• the nuclease variants contemplated in particular embodiments can be used to introduce a double-strand break in a target polynucleotide sequence, which may be repaired by non-homologous end joining (NHEJ) in the absence of a polynucleotide template, e.g., a donor repair template, or by homology directed repair (HDR), i.e., homologous recombination, in the presence of a donor repair template.
• NHEJ non-homologous end joining
• HDR homology directed repair
• Nuclease variants contemplated in certain embodiments can also be designed as nickases, which generate single- stranded DNA breaks that can be repaired using the cell ' s base-excision-repair (BER) machinery or homologous recombination in the presence of a donor repair template.
• NHEJ is an error-prone process that frequently results in the formation of small insertions and deletions that disrupt gene function.
• Homologous recombination requires homologous DNA as a template for repair and can be leveraged to create a limitless variety of modifications specified by the introduction of donor DNA containing the desired sequence at the target site, flanked on either side by sequences bearing homology to regions flanking the target site.
• the TCRa gene targeting homing endonuclease variants or megaTALs contemplated herein have improved binding site selectivity or specificity to the target site while retaining high catalytic activity compared to existing homing endonucleases or megaTALs that target the TCRa gene.
• the term "about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• in vivo refers generally to activities that take place inside an organism.
• cellular genomes are engineered, edited, or modified in vivo.
• measurable response may include an increase in engineered TCR or CAR expression, increase in HR or HDR efficiency, increase in binding site selectivity, increase in binding site specificity, increase in on-target binding, increases in immune effector cell expansion, activation, persistence, and/or an increase in cancer cell death killing ability, among others apparent from the understanding in the art and the description herein.
• a binding domain binds to a target site with a K a greater than or equal to about 10 6 M “1 , 10 7 M “1 , 10 8 M “1 , 10 9 M “1 , 10 10 M “1 , 10 11 M “1 , 10 12 M “1 , or 10 13 M “1 .
• "High affinity" binding domains refers to those binding domains with a K a of at least 10 7 M “1 , at least 10 8 M “1 , at least 10 9 M “1 , at least 10 10 M “1 , at least 10 11 M “1 , at least 10 12 M “1 , at least 10 13 M _1 , or greater.
• selectively binds or “selectively bound” or “selectively binding” or “selectively targets” and describe preferential binding of one molecule to a target molecule (on- target binding) in the presence of a plurality of off-target molecules.
• nuclease comprising one or more DNA binding domains and one or more DNA cleavage domains, wherein the nuclease has been designed and/or modified from a parental or naturally occurring nuclease, to bind and cleave a double-stranded DNA target sequence in exon 1 of the constant region of the human TCRa gene.
• LHEs include, but are not limited to I-AabMI, I-AaeMI, I- Anil, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMLV, I-CpaMV, I-CpaV, I-CraMI, I-Ej eMI, I-GpeMI, I-Gpil, I-GzeMI, I-GzeMII, I-GzeMIII, I-Hj eMI, I-Ltrll, I-Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I-Ncrll, I-Ncrl, I-NcrMI, I-OheMI, I-Onul, I-OsoMI, I- OsoMII, I-OsoMIII, I-OsoMIV, I-OheMI,
• a megaTAL comprises 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, 15.5, 16.5, 17.5, 18.5, 19.5, 20.5, 21.5, 22.5, 23.5, 24.5, 25.5, 26.5, 27.5, 28.5, 29.5, or 30.5 TALE DNA binding domain repeat units.
• a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 5.5-15.5 repeat units, more preferably 7.5-15.5 repeat units, more preferably 9.5-15.5 repeat units, and more preferably 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, or 15.5 repeat units.
• the NTD polypeptide comprises at least about 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or at least 140 amino acids N-terminal to the TALE DNA binding domain.
• a megaTAL contemplated herein comprises an NTD polypeptide of at least about amino acids +1 to +122 to at least about +1 to +137 of aXanthomonas TALE protein (0 is amino acid 1 of the most N- terminal repeat unit).
• a megaTAL contemplated herein comprises a fusion polypeptide comprising a TALE DNA binding domain engineered to bind a target sequence, a homing endonuclease reprogrammed to bind and cleave a target sequence, and optionally an NTD and/or CTD polypeptide, optionally joined to each other with one or more linker polypeptides contemplated elsewhere herein.
• a megaTAL comprising TALE DNA binding domain, and optionally an NTD and/or CTD polypeptide is fused to a linker polypeptide which is further fused to a homing endonuclease variant.
• the TALE DNA binding domain binds a DNA target sequence that is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides away from the target sequence bound by the DNA binding domain of the homing endonuclease variant.
• the megaTALs contemplated herein increase the specificity, selectivity, and efficiency of genome editing.
• DNA end-processing enzymes suitable for use in particular embodiments contemplated herein include, but are not limited to: 5 ' -3 ' exonucleases, 5 ' -3 ' alkaline exonucleases, 3 ' -5 ' exonucleases, 5 ' flap endonucleases, helicases, phosphatases, hydrolases and template-independent DNA polymerases.
• exonucleases suitable for use in particular embodiments contemplated herein include, but are not limited to: hExoI, Yeast Exol, E. coli Exol, hTREX2, mouse TREX2, rat TREX2, hTREXl, mouse TREXl, rat TREX1, and Rat TREXl.
• glycosylations acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
• isolated protein refers to in vitro synthesis, isolation, and/or purification of a peptide or polypeptide molecule from a cellular environment, and from association with other components of the cell, i.e., it is not significantly associated with in vivo substances.
• a polypeptide fragment can comprise an amino acid chain at least 5 to about 1700 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids long.
• an I-Onul variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or a deletion of the following 1, 2, 3, 4, or 5 C-terminal amino acids: R, G, S, F, V.
• an I-Onul variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or a deletion of the following 1 or 2 C-terminal amino acids: F, V.
• polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
• amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985,
• a fusion protein contemplated herein comprises nuclease variant; a linker or self-cleaving peptide; and an end-processing enzyme including but not limited to a 5'- 3 ' exonuclease, a 5 ' -3 ' alkaline exonuclease, and a 3 ' -5 ' exonuclease (e.g., Trex2).
• an end-processing enzyme including but not limited to a 5'- 3 ' exonuclease, a 5 ' -3 ' alkaline exonuclease, and a 3 ' -5 ' exonuclease (e.g., Trex2).
• the polypeptides of the fusion protein can be in any order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be produced by chemical synthetic methods or by chemical linkage between the two moieties or may generally be prepared using other standard techniques.
• Fusion polypeptides may optionally comprises a linker that can be used to link the one or more polypeptides or domains within a polypeptide.
• a peptide linker sequence may be employed to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structures so as to allow the polypeptide domains to exert their desired functions.
• Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques in the art.
• LRQKDGGGSERP SEQ ID NO: 39
• LRQKD(GGGS) 2 ERP SEQ ID NO: 40
• Fusion polypeptides may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein or between an endogenous open reading frame and a polypeptide encoded by a donor repair template.
• a polypeptide cleavage site can be put into any linker peptide sequence.
• Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).
• RNA(+) minus strand RNA
• RNA(-) minus strand RNA
• crRNA crRNA
• single guide RNA sgRNA
• synthetic RNA synthetic mRNA
• genomic DNA gDNA
• PCR amplified DNA PCR amplified DNA
• polynucleotides may be codon-optimized.
• codon-optimized refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and/or activity of the polypeptide.
• sequence identity or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by- nucleotide basis or an amino acid-by -amino acid basis over a window of comparison.
• a polynucleotide comprises an mRNA encoding a polypeptide contemplated herein including, but not limited to, a homing endonuclease variant, a megaTAL, and an end-processing enzyme.
• the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.
• mRNAs comprise a 5 ' cap wherein the cap is a CapO structure (CapO structures lack a 2 ' -0-methyl residue of the ribose attached to bases 1 and 2), a Capl structure (Capl structures have a 2 ' -0-methyl residue at base 2), or a Cap2 structure (Cap2 structures have a 2 ' -0-methyl residue attached to both bases 2 and 3).
• the cap is a CapO structure
• CapO structures lack a 2 ' -0-methyl residue of the ribose attached to bases 1 and 2
• a Capl structure Capl structures have a 2 ' -0-methyl residue at base 2
• a Cap2 structure Cap2 structures have a 2 ' -0-methyl residue attached to both bases 2 and 3).
• an mRNA comprises one or more pseudouridines, one or more 5- methyl-cytosines, and/or one or more 5-methyl-cytidines.
• an mRNA comprises one or more 5-methyl-cytidines.
• an mRNA comprises one or more 5-methyl-cytosines.
• the length of the poly(A) tail is at least about 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 202, 203, 205, 206, 207, 208, 209,
• the length of the poly(A) tail is about 10 to about 500 adenine nucleotides, about 50 to about 500 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 250 to about 500 adenine nucleotides, about 300 to about 500 adenine nucleotides, about 50 to about 450 adenine nucleotides, about 50 to about 400 adenine nucleotides, about 50 to about 350 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 100 to about 450 adenine nucleotides, about 100 to about 400 adenine nucleotides, about 100 to about 350 adenine nucleotides, about 100 to about 300 adenine nucleotides, about 150 to about 500 adenine nucleot
• complementary and complementarity refer to polynucleotides ⁇ i.e., a sequence of nucleotides) related by the base-pairing rules.
• the complementary strand of the DNA sequence 5 ⁇ G T C A T G 3 ' is 3 ' T C A G T A C 5 ' .
• the latter sequence is often written as the reverse complement with the 5 ' end on the left and the 3 ' end on the right, 5 C A T G A C T 3 ' .
• a sequence that is equal to its reverse complement is said to be a palindromic sequence.
• Complementarity can be "partial,” in which only some of the nucleic acids ' bases are matched according to the base pairing rules. Or, there can be “complete” or “total” complementarity between the nucleic acids.
• vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses.
• artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC)
• bacteriophages such as lambda phage or Ml 3 phage
• animal viruses include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses.
• expression vectors include, but are not limited to pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST TM , pLenti6/V5- DESTTM, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
• coding sequences of polypeptides disclosed herein can be ligated into such expression vectors for the expression of the polypeptides in mammalian cells.
• the vector is an episomal vector or a vector that is maintained extrachromosomally.
• episomal vector refers to a vector that is able to replicate without integration into host ' s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally.
• an "internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene. See, e.g., Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES generally employed by those of skill in the art include those described in U.S. Pat. No. 6,692,736.
• IRES VEGF IRES
• Picornaviridae Dicistroviridae and Flaviviridae species
• HCV Friend murine leukemia virus
• MoMLV Moloney murine leukemia virus
• the core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage-polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5 ' cleavage product.
• the core poly(A) sequence is a synthetic poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA).
• non-viral vectors include, but are not limited to plasmids
• polynucleotide delivery systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc.
• Lipofection reagents are sold commercially (e.g.,
• autologous/autogeneic self or non-autologous
• non-self e.g., allogeneic, syngeneic or xenogeneic
• autologous refers to cells from the same subject.
• Allogeneic refers to cells of the same species that differ genetically to the cell in comparison.
• Syngeneic refers to cells of a different subject that are genetically identical to the cell in comparison.
• Xenogeneic refers to cells of a different species to the cell in comparison.
• the cells are obtained from a mammalian subject.
• the cells are obtained from a primate subject, optionally a non-human primate.
• the cells are obtained from a human subject.
• Protent T cells and “young T cells,” are used interchangeably in particular embodiments and refer to T cell phenotypes wherein the T cell is capable of proliferation and a concomitant decrease in differentiation.
• the young T cell has the phenotype of a "naive T cell.”
• young T cells comprise one or more of, or all of the following biological markers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, and CD38.
• young T cells comprise one or more of, or all of the following biological markers: CD62L, CD127, CD197, and CD38.
• the young T cells lack expression of CD57, CD244, CD160, PD-1, CTLA4, and LAG3.
• T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
• a population of cells comprises immune effector cells or T cells comprising a homing endonuclease variant or megaTAL that targets exon 1 of the constant region of the TCRa gene as contemplated herein.
• a population of cells comprises immune effector cells or T cells comprising a homing endonuclease variant polypeptide or megaTAL polypeptide that targets exon 1 of the constant region of the TCRa gene as contemplated herein.
• the compositions contemplated herein comprise a donor repair template.
• the composition may be delivered to a cell that expresses or will express nuclease variant, and optionally an end-processing enzyme.
• the composition may be delivered to a cell that expresses or will express a homing endonuclease variant or megaTAL, and optionally a 3 '-5' exonuclease.
• Expression of the gene editing enzymes in the presence of the donor repair template can be used to generate a genome edited cell or population of genome edited cells by HDR.
• buffers refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH.
• buffers contemplated herein include, but are not limited to, Dulbecco ' s phosphate buffered saline (PBS), Ringer ' s solution, 5% dextrose in water (D5W), normal/physiologic saline (0.9% NaCl).
• Serum-free medium has several advantages over serum containing medium, including a simplified and better defined composition, a reduced degree of contaminants, elimination of a potential source of infectious agents, and lower cost.
• the serum-free medium is animal-free, and may optionally be protein-free.
• the medium may contain biopharmaceutically acceptable recombinant proteins.
• Animal-free medium refers to medium wherein the components are derived from non-animal sources. Recombinant proteins replace native animal proteins in animal- free medium and the nutrients are obtained from synthetic, plant or microbial sources.
• Protein-free in contrast, is defined as substantially free of protein.
• compositions comprising genome edited T cells are formulated in a cryopreservation medium.
• cryopreservation media with cryopreservation agents may be used to maintain a high cell viability outcome post-thaw.
• cryopreservation media used in particular compositions includes, but is not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

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