WO2017023801A1 - Intracellular genomic transplant and methods of therapy - Google Patents

Intracellular genomic transplant and methods of therapy Download PDF

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Publication number
WO2017023801A1
WO2017023801A1 PCT/US2016/044856 US2016044856W WO2017023801A1 WO 2017023801 A1 WO2017023801 A1 WO 2017023801A1 US 2016044856 W US2016044856 W US 2016044856W WO 2017023801 A1 WO2017023801 A1 WO 2017023801A1
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cell
receptor
gene
sequence
cells
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PCT/US2016/044856
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English (en)
French (fr)
Inventor
Branden MORIARITY
Beau WEBBER
R. Scott Mcivor
Modassir CHOUDHRY
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University of Minnesota Twin Cities
Intima Bioscience Inc
University of Minnesota System
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University of Minnesota Twin Cities
Intima Bioscience Inc
University of Minnesota System
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Definitions

  • compositions and methods herein can be used for the identification of cancer-specific T Cell Receptors (TCRs) that recognize unique immunogenic mutations in a patient's cancer and to treat any type of cancer within a patient. Insertion of these transgenes encoding the cancer-specific TCR into T cells using non-viral (e.g. , CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega- TAL) methods are innovative approaches that opens new opportunities for extending immunotherapy to many cancer types.
  • non-viral e.g. , CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega- TAL
  • engineered cells comprising at least one gene disruption and at least one non- virally integrated T cell receptor (TCR) sequence, where the gene can be disrupted by the non-virally integrated TCR sequence.
  • the gene can be a checkpoint gene, for example, the gene can be an immune checkpoint gene.
  • the gene can be adenosine A2a receptor (ADORA), CD276, V- set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO l), killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte- activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244), cytokine inducible SH2 -containing protein (CISH), hypoxanthine
  • ADORA adenosine A2a receptor
  • CD276 V- set domain containing T cell activation inhibitor 1
  • BTLA B and T lymphocyte associated
  • phosphoribosyltransferase 1 HPRT
  • AAVS SITE adeno-associated virus integration site
  • C-C motif chemokine receptor 5 (gene/pseudogene) (CCR5).
  • the gene can be PD-1.
  • the engineered cell can comprises a single TCR sequence.
  • the TCR sequence can comprises an engineered TCR sequence.
  • the TCR sequence can comprise two or more chains.
  • the two or more chains can comprise at least one alpha chain.
  • the two or more chains can comprise at least one beta chain.
  • the TCR sequence can comprises an extracellular region, a transmembrane region, and an intracellular region.
  • the TCR sequence can produce a functional TCR.
  • the TCR sequence can recognizes antigen.
  • the TCR sequence can recognize antigen in the context of a major
  • the MHC can be class I.
  • the MHC can be HLA-A02.
  • the MHC can be class II.
  • the TCR can bind to a mutation.
  • the mutation that the TCR binds to can be identified by whole-exomic sequencing.
  • the TCR can bind to cancer cells.
  • the engineered cell can be a primary cell.
  • the engineered cell can be an immune cell.
  • the engineered cell can be a T cell, a stem cell, or a progenitor cell.
  • the engineered cell can be a hematopoietic progenitor cell.
  • the engineered cell can be a human cell.
  • the engineered cell can be selected.
  • the engineered cell can be expanded ex vivo.
  • the engineered cell can be expanded in vivo.
  • the engineered cell can be CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), or IL-7Ra(+).
  • the engineered cell can be autologous to a subject in need thereof.
  • the engineered cell can be non-autologous to a subject in need thereof.
  • the engineered cell can be a good manufacturing practices (GMP) compatible reagent.
  • the engineered cell can be a part of a combination therapy to treat cancer, infections, autoimmune disorders, or graft-versus-host disease (GVHD) in a subject in need thereof.
  • GMP good manufacturing practices
  • GVHD graft-versus-host disease
  • TCR T cell receptor
  • recombination arms introducing into a cell one or more polynucleic acids comprising at least one exogenous T cell receptor (TCR) sequence flanked by recombination arms; and b) contacting the at least one exogenous TCR sequence with a double stranded break region that comprises a gene.
  • the recombination arms can be complementary to a portion of the gene.
  • the gene can be adenosine A2a receptor, CD276, V- set domain containing T cell activation inhibitor 1, B and T lymphocyte associated, cytotoxic T- lymphocyte-associated protein 4, indoleamine 2,3-dioxygenase 1, killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1, lymphocyte-activation gene 3, programmed cell death 1 (PD-1), hepatitis A virus cellular receptor 2, V-domain immunoglobulin suppressor of T-cell activation, or natural killer cell receptor 2B4.
  • the gene can be PD-1.
  • the gene can be a checkpoint gene.
  • the checkpoint gene can be an immune checkpoint gene.
  • the double strand break region can be repaired by insertion of the at least one exogenous TCR
  • the insertion of the at least one exogenous TCR sequence can comprise disruption of the at least one gene.
  • the insertion of the at least one exogenous TCR sequence can be assisted by a homologous recombination (HR) enhancer.
  • the enhancer can be derived from a viral protein.
  • the enhancer can be E1B55K, E4orf6, Scr7, or L755507.
  • the enhancer can be a chemical inhibitor.
  • the enhancer inhibits Ligase IV.
  • the enhancer can facilitate insertion of the TCR sequence.
  • the insertion can comprise homology directed repair.
  • the double strand break region can be created by CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega-TAL.
  • the double strand break region can be created by CRISPR.
  • CRISPR can be multiplexed.
  • multiplexing can be performed by adding at least 2 guide RNAs. The TCR sequence can be inserted near the double strand break region.
  • the polynucleic acid can be RNA.
  • the RNA can be mRNA.
  • the cell can be contacted with reverse transcriptase (RT).
  • RT reverse transcriptase
  • the cell can be contacted with primers that are complementary to the polynucleic acid.
  • the RT transcribes the mRNA into a first ssDNA template.
  • the RT transcribes the first ssDNA template into a second dsDNA template.
  • transcribing can be performed in situ.
  • the ssDNA or dsDNA can comprise the at least one exogenous TCR sequence.
  • primer sequences can be used to determine the presense of an RT.
  • a Reverse Transcriptase (RT) reporter forward primer can be AAC GTG CTG GTT GTT GTG CTG (SEQ ID NO 180). In other cases, a Reverse Transcriptase (RT) reporter reverse primer can be used.
  • An RT reporter reverse primer can be AAA GTG GTG GTA GAA TAG GCT C (SEQ ID NO 181).
  • non-viral introduction can comprise electroporation or nucleofection.
  • a polynucleic acid can be co-delivered with at least one modifier that alters cellular response to a polynucleic acid. At least one modifier can reduce cellular toxicity.
  • a modifier can comprise abPan Caspase Inhibitor Z-VAD-FMK or BX795.
  • the invention can comprise a primary cell.
  • the primary cell can be an immune cell.
  • the immune cell can be a T cell, a stem cell, or a progenitor cell.
  • the method can comprise a progenitor cell.
  • a progenitor cell is a hematopoietic progenitor cell.
  • the cell is a human cell.
  • the method can be good manufacturing practices (GMP) compatible.
  • a subject in need thereof receives treatment comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an engineered cell.
  • a pharmaceutical composition can be administered intravenously.
  • a pharmaceutical composition can be administered locally.
  • a method can further comprise administering one more or more additional therapies.
  • the one or more additional therapies can comprise transplantation.
  • the one or more additional therapies can comprise immunotherapy.
  • the engineered cell can be autologous to the subject. In some cases, the engineered cell can be allogenic to the subject.
  • polynucleic acids comprising at least one exogenous T cell receptor (TCR) sequence flanked by at least two recombination arms having a sequence complementary to a genomic sequence that can be adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), cytotoxic T-lymphocyte- associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDOl), killer cell immunoglobulin- like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte-activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244), cytokine inducible SH2-containing protein (CISH), hypox
  • TCR exogenous T
  • the polynucleic acid sequence can be complementary to a genomic sequence that can be a partial sequence. In some cases, binding of the recombination arms to the sequence complementary to a genomic sequence inserts the exogenous TCR sequence. In some cases, binding of the recombination arms to the sequence complementary to a genomic sequence repairs a double strand break. In some cases, the genomic sequence comprises a coding sequence. In some cases, the genomic sequence comprises a non-coding sequence. In some cases, the genomic sequence comprises one or more genes. Insertion of the exogenous TCR sequence can disrupt one or more genes. In some cases, the genomic sequence can be PD-1.
  • the polynucleic acid can be a plasmid vector.
  • the plasmid vector can comprise a
  • the promotor can be constitutive. In some cases, the promoter can be inducible. The promoter can be CMV, U6, MND, or EFla. In some cases, the promoter can be adjacent to the exogenous TCR sequence. In some cases, the plasmid vector further comprises a splicing acceptor. In some cases, the splicing acceptor can be adjacent to the exogenous TCR sequence.
  • An MND promoter can be a synthetic promoter that contains a U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer.
  • the plasmid vector further comprises an "ATG" sequence.
  • the "ATG” sequence can be adjacent to the TCR sequence.
  • the TCR sequence encodes for a fusion protein.
  • the TCR sequence can be within a multicistronic vector.
  • the polynucleic acid comprises an exogenous promotor, an endogenous promoter via splicing, and/or an endogenous promoter via in frame translation.
  • the plasmid can be modified.
  • the modification can comprise demethylation, addition of CpG methylation, removal of bacterial methylation, and addition of mammalian methylation.
  • the TCR sequence can be an engineered TCR sequence.
  • the polynucleic acid can be designed to be delivered to a cell by non-viral techniques.
  • the polynucleic acid can be a good manufacturing practices (GMP) compatible reagent.
  • GMP good manufacturing practices
  • HDR homology directed repair
  • the method can comprise generating a double stranded break.
  • the double strand break can be performed by CRISPR, TALEN, transposon-based, ZEN, meganuclease, and Mega-TAL.
  • the double strand break can be performed by CRISPR.
  • the HDR of c) repairs the double strand break.
  • the CRISPR can be multiplexed with at least two (2) guide RNAs.
  • the polynucleic acid can be DNA. In some cases, the polynucleic acid can be cDNA. In some cases, the polynucleic acid can be single stranded.
  • the RT transcribes the mRNA into a first ssDNA template. In some cases, the
  • polynucleic acid can be double stranded.
  • the RT transcribes the mRNA into a second dsDNA template in situ.
  • the mRNA or polynucleic acid can comprises at least one TCR sequence.
  • the TCR sequence comprises at least two flanking recombination arms having a sequence complementary to a genomic region.
  • the TCR sequence can be used in HDR of c).
  • the TCR sequence can be used in HDR of c) and further comprises binding of the recombination arms to a complementary portion of the genome of the cell.
  • the TCR sequence can be used in HDR of c) and further comprises binding of the recombination arms to a complementary portion of the genome of the cell and further comprises insertion of the TCR sequence.
  • HDR between the genome of the cell and of the polynucleic acid disrupts one or more genes.
  • One or more genes can comprise an immune checkpoint gene.
  • one or more genes comprise adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), cytotoxic T-lymphocyte- associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDOl), killer cell immunoglobulin- like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte-activation gene 3
  • ADORA adenosine A2a receptor
  • VTCN1 V-set domain containing T cell activation inhibitor 1
  • BTLA B and T lymphocyte associated
  • CTLA4 cytotoxic T-lymphocyte- associated protein 4
  • IDOl indoleamine 2,3-dioxygenase 1
  • KIR3DL1 lymphocyte-activation gene 3
  • LAG3 programmed cell death 1
  • HAVCR2 hepatitis A virus cellular receptor 2
  • VISTA V-domain immunoglobulin suppressor of T-cell activation
  • CD244 natural killer cell receptor 2B4
  • CISH cytokine inducible SH2-containing protein
  • HPRT adeno-associated virus integration site
  • AAVS SITE E.G. AAVS 1, AAVS2, ETC.
  • C-C motif gene/pseudogene
  • one or more genes comprise PD-1.
  • one or more genes comprise a TCR.
  • HDR between the genome of the cell and of the polynucleic acid can be assisted by one or more homologous recombination (HR) enhancers.
  • the one or more enhancers can comprise a viral protein.
  • one or more enhancers comprise E1B55K, E4orf6, Scr7, and/or L755507.
  • the enhancer comprises a chemical inhibitor.
  • the enhancer inhibits Ligase IV.
  • the enhancer facilitates insertion of the polynucleic acid into the genome of the cell.
  • the enhancer can prevent non homologous end joining (NHEJ).
  • NHEJ non homologous end joining
  • the polynucleic acid can be inserted at or near the double strand break.
  • the mRNA, reverse transcriptase, primer, HR enhancer, and CRISPR are contacted with the cell.
  • the polynucleic acid, CRISPR, and HR enhancer are contacted with the cell.
  • the cell can be a primary cell.
  • the cell can be an immune cell.
  • the cell can be a T-cell, a stem cell, or a progenitor cell.
  • the cell is a T cell.
  • the cell is a progenitor cell.
  • the cell is a hematopoietic progenitor cell.
  • the cell can be human.
  • the T cell can be autologous.
  • the T cell can be non -autologous.
  • the method can be good manufacturing practices (GMP) compatible.
  • altering one or more cellular responses comprises modifying DNA-dependent activator of IFN regulatory factors (DAI), IFN inducible protein 16 (IFI16), DEAD box polypeptide 41 (DDX41), absent in melanoma 2 (AIM2), DNA-dependent protein kinase, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), stimulator of IFN genes (STING), TANK-binding kinase (TBK1), interleukin-1 ⁇ (IL- ⁇ ), MREl 1, meiotic recombination 11, Trexl, cysteine protease with aspartate specificity (Caspase-1), three prime repair exonuclease, DNA-dependent activator of IRFs (DAI), IFI
  • one or more compounds alter one or more cellular responses.
  • One or more compounds can comprise an inhibitor.
  • One or more compounds can comprise an activator.
  • one or more compounds comprise Pan Caspase Inhibitor, Z- VAD-FMK, and/or Z-VAD-FMK.
  • one or more compounds are modified.
  • One or more compounds can prevent cellular apoptosis and pyropoptosis.
  • one or more compounds can inhibit Caspase-1 from cleaving proIL- ⁇ and proIL-18.
  • one or more compounds can modulate activity of an apoptosis-associated speck-like protein containing a CARD (ASC).
  • ASC apoptosis-associated speck-like protein containing a CARD
  • One or more compounds can modulate a cGAS-STING pathway.
  • One or more compounds can prevent expression of type I interferons.
  • one or more compounds can comprise two or more compounds.
  • the compound can be good manufacturing practices (GMP) compatible.
  • the compound can be contacted with the cell prior to contacting the cell with the one or more exogenous engineered polynucleic acids.
  • the method can further comprise contacting the cell with one or more homologous recombination (HR) enhancers.
  • HR homologous recombination
  • the method can further comprise selecting the cell. In some cases, the method can further comprise expanding the cell. In some cases, the method produces a GMP compatible cellular therapy.
  • the one or more signaling modifier compound alters a cytosolic DNA-sensing pathway.
  • the one or more signaling modifier compound alters DNA -dependent activator of IFN regulatory factors (DAI), IFN inducible protein 16 (IFI16), DEAD box polypeptide 41 (DDX41), absent in melanoma 2 (AIM2), DNA -dependent protein kinase, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), stimulator of IFN genes (STING), TANK-binding kinase (TBK1), interleukin-1 ⁇ (IL- ⁇ ), MRE11, meiotic recombination 11, Trexl, cysteine protease with aspartate specificity (Caspase-1), three prime repair exonuclease, DNA- dependent activator of IRFs (DAI), IFI16, DDX41, DNA-dependent protein kinase (DNA-PK), meiotic recombination 11 homolog A (MRE11), and/or IFN regulatory factor (I), IFN
  • the one or more signaling modifier compound comprises an inhibitor. In some cases, the one or more signaling modifier compound comprises an activator.
  • the one or more signaling modifier compound can comprise Pan Caspase Inhibitor, Z-VAD-FMK, and/or Z-VAD-FMK.
  • the one or more signaling modifier compound can be modified.
  • the one or more signaling modifier compound can be modified.
  • the signaling modifier compound can prevent cellular apoptosis and pyropoptosis.
  • the one or more signaling modifier compound inhibits Caspase-1 from cleaving proIL- ⁇ and proIL-18.
  • the one or more signaling modifier compound can modulate activity of apoptosis-associated speck-like protein containing a CARD (ASC).
  • the one or more signaling modifier compound modulates a cGAS-STING pathway.
  • the one or more signaling modifier compound can prevent expression of type I interferons.
  • the one or more signaling modifier compound can comprise two or more compounds.
  • the one or more signaling modifier compound can be contacted with the cell prior to contacting the cell with the one or more exogenous engineered polynucleic acids.
  • the method can further comprise contacting the cell with one or more homologous recombination (HR) enhancers.
  • the cell is a primary cell.
  • the cell is an immune cell.
  • the cell is a T cell, a stem cell, or a progenitor cell.
  • the invention can comprise a progenitor cell.
  • the cell can be a hematopoietic progenitor cell.
  • the cell can be a human cell.
  • unmethylated polynucleic acids comprising at least one engineered antigen receptor flanked by at least two recombination arms complementary to at least one genomic region.
  • the polynucleic acid can be modified.
  • the modification can be demethylation, addition of CpG methylation, removal of bacterial methylation, and/or addition of mammalian methylation.
  • the polynucleic acid can be capable of undergoing homologous recombination.
  • the recombination arms bind a complementary genomic region.
  • the antigen receptor comprises a TCR or a chimeric antigen receptor (CAR).
  • mammalian methylated polynucleic acids comprising at least one
  • the polynucleic acid can be further modified.
  • Modification can be demethylation, addition of CpG methylation, removal of bacterial methylation, and/or addition of mammalian methylation.
  • the polynucleic acid can be capable of undergoing homologous recombination.
  • the mammalian methylated polynucleic acid can further comprise recombination arms that bind to at least one complementary genomic region. In some cases, the recombination arms bind a complementary genomic region.
  • the mammalian methylated polynucleic can comprise an antigen receptor comprising a TCR or a chimeric antigen receptor (CAR).
  • composition for reducing cellular toxicity comprising a caspase
  • a caspase modulator can alter a cytosolic DNA- sensing pathway.
  • a cGAS-STING pathway modulator can alter a cytosolic DNA-sensing pathway.
  • the cytosolic DNA-sensing pathway can comprise caspase- 1.
  • the caspase modulator can be a caspase inhibitor.
  • the caspase modulator can inhibit caspase- 1 from cleaving proIL- ⁇ and proIL-18.
  • the cytosolic DNA-sensing pathway can comprise a DNA-dependent activator of IFN regulatory factors (DAI), IFN inducible protein 16 (IFI16), DEAD box polypeptide 41 (DDX41), absent in melanoma 2 (AIM2), DNA-dependent protein kinase, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), stimulator of IFN genes (STING), TANK-binding kinase (TBK1), interleukin-1 ⁇ (IL- ⁇ ), MREl 1, meiotic recombination 11, Trexl, cysteine protease with aspartate specificity (Caspase-1), three prime repair exonuclease, DNA-dependent activator of IRFs (DAI),
  • DAI DNA-dependent activator of IFN regulatory factors
  • IFI16 IFN inducible protein 16
  • DDX41 DEAD box polypeptide 41
  • AIM2 melanom
  • the cGAS-STING pathway modulator can be a cGAS-STING pathway inhibitor.
  • the cGAS-STING pathway inhibitor can comprise a Pan Caspase
  • the composition can prevent cellular apoptosis and pyropoptosis.
  • the composition can prevent expression of type I interferons.
  • the composition can reduce cellular toxicity comprising a modified caspase modulator.
  • the composition can reduce cellular toxicity comprising a modified cGAS-STING pathway modulator.
  • the modification can comprise deuteration, lipidization, glycosylation, alkylation,
  • the modification can improve activity of the modified caspase modulator and the modified cGAS-STING pathway modulator.
  • the activity can increase by about or by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 500, 750, or 1000% or more compared to a non-modified caspase modulator or non-modified cGAS-STING pathway modulator.
  • the activity can increase by at least about or by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 500, 750, or 1000% or more compared to a non -modified caspase modulator or non -modified cGAS-STING pathway modulator.
  • the activity can increase by at least about or by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 500, 750, or 1000% and up to 100% compared to a non-modified caspase modulator or non-modified cGAS-STING pathway modulator.
  • the composition is introduced to the cell. In some cases, the composition can prevent toxicity in a cell. In some cases, the cell is further contacted with the polynucleic acid.
  • a method for making an engineered cell comprising; introducing into a cell a guiding polynucleotide comprising a spacer region that is complementary to a target nucleic acid in a genomic region of the cell; a nuclease that is guided by the guiding polynucleotide; and a polynucleotide encoding an exogenous T cell receptor; site-specifically cleaving the target nucleic acid inside the cell by the nuclease guided by the guiding polynucleotide; and inserting the polynucleotide encoding the exogenous T cell receptor into the genomic region of the cell at the cleavage site.
  • the nuclease can be Cas9.
  • the guiding polynucleotide can be a single guiding polynucleotide.
  • the guiding polynucleotide can be R A.
  • the target nucleic acid can be DNA.
  • the spacer region can be between 10-30 nucleotides in length.
  • the nuclease can produce a double stranded break in the target nucleic acid.
  • the guiding polynucleotide can be introduced into a cell by electroporation.
  • a guide nucleic acid can be introduced into a cell by nucleofection.
  • a nuclease can also be introduced into a cell by a delivery vector.
  • a polynucleotide encoding an exogenous T cell receptor can further comprise a promoter sequence.
  • a promoter sequence can be a PKG or an MND promoter.
  • An exogenous T cell receptor can be inserted by homologous recombination.
  • a guiding polynucletotide and a nuclease can form a nucleoprotein complex.
  • cleaving a target nucleic acid can remove a genomic nucleic acid
  • polynucleotide encoding an exogenous T cell receptor can further comprise a first recombination arm and a second recombination arm.
  • a first recombination arm can comprise a first sequence that is identical to a first portion of a target nucleic acid and a second recombination arm can comprise a second sequence that is identical to a second portion of a target nucleic acid.
  • a first recombination arm can comprise a first sequence that is identical to a first portion adjacent to a target nucleic acid and a second recombination arm can comprise a second sequence that is identical to a second portion adjacent to a target nucleic acid.
  • a target nucleic acid can be within a gene.
  • a gene can be selected from adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), cytotoxic T-lymphocyte- associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDOl), killer cell immunoglobulin- like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte-activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244), cytokine inducible SH2-containing protein (CISH), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS SITE (E.G.
  • ADORA adenosine A
  • a gene can be PD-1.
  • a gene can be a checkpoint gene.
  • a checkpoint gene can be an immune checkpoint gene.
  • insertion of an exogenous TCR sequence at a cleavage site can result in disruption of a gene.
  • a target nucleic acid can be within an intergenic site.
  • An exogenous T cell receptor can be expressed in a cell.
  • An engineered cell can be introduced into an organism. Engineered cells can be expanded ex vivo.
  • NHEJ non-homologous end joining
  • Suppressing NHEJ in a cell can comprise inhibiting Ligase IV.
  • Suppressing NHEJ in a cell can also comprise introducing a homologous recombination (HR) enhancer.
  • HR homologous recombination
  • An enhancer can be derived from a viral protein.
  • An enhancer can be E1B55K, E4orf6, Scr7, or L755507.
  • Suppressing NHEJ in a cell can facilitate insertion of a polynucleotide encoding an exogenous TCR at a cleavage site by homologous recombination.
  • a modifier can be Pan Caspase Inhibitor Z-VADFMK and/or BX795.
  • a cell can be a T cell.
  • a cell can be a mammalian cell.
  • a cell can be a primary cell.
  • a primary cell can be an immune cell.
  • a cell can be a stem cell, or a progenitor cell.
  • a cell is a progenitor cell.
  • a progenitor cell can be a hematopoietic progenitor cell.
  • a cell can be a human cell.
  • Disclosed herein can also be a composition comprising an engineered cell.
  • An engineered cell can be administered to a subject in a therapeutically effective amount.
  • Administration of an engineered cell can produce a therapeutic outcome in a subject, wherein a therapeutic outcome is modulated by an exogenous TCR.
  • a protospacer adjacent motif sequence can be recognized by a CRISPR endonuclease.
  • An endonuclease can be a Cas protein.
  • a Cas protein can be selected from a list comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl or Csxl2), CaslO, Csyl , Csy2, Csy3, Cse l, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Cpfl, c2cl, c2c3, Cas9HiFi, homologues thereof or modified versions thereof.
  • a CRISPR endonuclease can be Cas9.
  • a Cas9 of the present invention can recognize a PAM sequence that may be 5' NGG 3'.
  • Disclosed herein can be at least one exogenous receptor that can disrupt at least one gene.
  • a gene can be a checkpoint gene.
  • a checkpoint gene can be selected from adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3- dioxygenase 1 (IDO l), killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte -activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244), cytokine inducible SH2 -containing protein (CISH), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS SITE (E.G. AAVS1, AAVS
  • CCR5 gene/pseudogene
  • a gene can comprise a protospacer.
  • a protospacer can be disrupted by insertion of an exogenous receptor sequence.
  • at least one exogenous receptor sequence can be an immune receptor sequence.
  • An immune receptor sequence can be selected from a list comprising a T cell receptor (TCR) sequence, a B cell receptor (BCR) sequence, or a chimeric antigen receptor (CAR) sequence.
  • TCR T cell receptor
  • BCR B cell receptor
  • CAR chimeric antigen receptor
  • a TCR sequence can comprise two or more chains. Two or more chains can comprise at least one alpha chain in the present invention. Two or more chains can also comprise at least one beta chain.
  • a TCR sequence can comprise an extracellular region, a transmembrane region, and an intracellular region.
  • a TCR sequence can produce a functional TCR.
  • a TCR sequence can recognize antigen.
  • a TCR sequence can recognize antigen in the context of major histocompatibility complex (MHC).
  • MHC can be class I.
  • MHC can be HLA-A02.
  • MHC can be class II.
  • a mutation can be an exogenous receptor that can bind to a mutation.
  • a mutation can be
  • a cell of the present invention can be a primary cell.
  • a primary cell can be an immune cell.
  • a cell can be a T cell, a stem cell, or a progenitor cell.
  • a cell can be a progenitor cell.
  • a progenitor cell can be a hematopoietic progenitor cell.
  • a cell of the present invention can be a human cell.
  • a cell can be selected.
  • a cell can be expanded ex vivo.
  • a cell can be expanded in vivo.
  • a cell can also be CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), IL-7Ra(+), or combinations thereof.
  • a cell of the present invention can be a cell that may be autologous to a subject in need thereof.
  • a cell can also be non-autologous to a subject in need thereof.
  • a cell can be a good manufacturing practices (GMP) compatible reagent.
  • GMP good manufacturing practices
  • a cell can be part of a combination therapy to treat cancer, infections, autoimmune disorders, or graft-versus-host disease (GVHD) in a subject in need thereof.
  • a cell of the present invention can be administered of a subject in need thereof as a
  • compositions comprising at least one guide RNA that binds to an endogenous cytokine inducible SH2 -containing (CISH) gene and a secondary guide RNA that binds to an endogenous gene selected from the group consisting of adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3- dioxygenase 1 (IDO l), killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte -activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD24
  • ADORA adenosine A2a
  • Disclosed herein can be an engineered cell with a disruption in an endogenous cytokine inducible
  • An endogenous gene can be selected from the group consisting of adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3- dioxygenase 1 (IDO l), killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte -activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS SITE
  • ADORA adenosine A2a receptor
  • VTCN1 T cell
  • a cell of the present invention can further comprise an exogenous receptor.
  • exogenous receptor can be selected from a group comprising a T cell receptor (TCR), Chimeric Antigen Receptor (CAR), or B cell receptor (BCR).
  • An exogenous receptor can binds to a mutation. A mutation can be identified by whole-exomic sequencing.
  • An exogenous receptor can bind to cancer cells.
  • An engineered cell can be a primary cell. A primary cell can be an immune cell.
  • a cell can be a T cell, a stem cell, or a progenitor cell.
  • a cell can be a progenitor cell.
  • a progenitor cell can be a hematopoietic progenitor cell.
  • a cell of the present invention can be a human cell.
  • a genetically modified immune cell comprising a lymphocyte, wherein a
  • lymphocyte is derived from a human subject; a polynucleic acid-targeting polynucleic acid, wherein a polynucleic acid-targeting polynucleic acid is engineered to hybridize to a specific region of a target gene in a genome of a lymphocyte; a nuclease, wherein a nuclease is capable of associating with a polynucleic acid-targeting polynucleic acid to form a nucleoprotein complex, wherein a nucleoprotein complex can be capable of generating a targeted double-strand break in a target gene in a genome of a lymphocyte; and a target polynucleic acid, wherein a target polynucleic acid can be genomic DNA comprising a double-strand break in a target gene, wherein a double-strand break in a target gene results in disruption of a target gene function and wherein a disruption of a target gene function occurs with at least 60% efficiency when a nucleoprotein complex can be contacted
  • a guide RNA contains a region of 17 to 22 nucleotides that is substantially complementary to a region in a target gene; cleaving a target gene, wherein a target gene can be PD-1 and wherein a knock out event occurs in at least 30% of primary T cells when a population of primary T cells are contacted with a Cas9 nuclease and a guide RNA; and disrupting a checkpoint inhibitor in a T cell.
  • a method of treating a subject in need thereof comprising collecting lymphocyte cells from a human; genetically modifying lymphocyte cells ex vivo by contacting a ribonuclease capable of knocking out PD-1 protein function by inducing a double strand break in a specific target region of genomic DNA in a lymphocyte cell, wherein a target region of genomic DNA in a lymphocyte is within a PD-1 gene and a double strand break occurs in a target region of genomic DNA that is 3 ' to a region of a target DNA that is capable of hybridizing to at least 15 nucleotides of a ribonuclease and is 5' to a region of a target DNA that contains a protospacer adjacent motif, expanding a population of genetically modified lymphocytes that have a knockout of PD-1 protein to generate a population of PD-1 knockout T cells; administering to a subject a population of PD-1 knockout T cells, wherein PD-1 knockout
  • the present disclosure provides methods of making genetically modified cells
  • the method comprises introducing into the one or more cells a first nucleic acid.
  • the method comprises a first nucleic acid, and the first nucleic acid comprises a first transgene encoding at least one anti-DNA sensing protein.
  • the method comprises at least one DNA sensing pathway, and the at least one DNA sensing pathway is disrupted within the one or more cells by at least one anti-DNA sensing protein.
  • the method comprises introducing into the one or more cells a second nucleic acid.
  • the method comprises a second nucleic acid, and the second nucleic acid comprises a second transgene encoding an engineered T-cell receptor (TCR.
  • TCR engineered T-cell receptor
  • the method comprises at least one endogenous immunological checkpoint gene, and the at least one endogenous immunological checkpoint gene is disrupted within the one or more cells by an insertion of the second transgene.
  • the method comprises the disruption of the at least one DNA sensing pathway reduces cytotoxicity induced by the second transgene, thereby maintaining or increasing viability of the one or more cells.
  • the method comprises one or more cells, and the one or more cells are immune cells.
  • the method comprises one or more cells, and the one or more cells are T cells, naive T cells, CD4+ cells, CD 8+ cells, stem cells, induced pluripotent stem cells, progenitor cells, hematopoetic cells, primary cells or any combination thereof.
  • the method comprises a first nucleic acid, and the first nucleic acid is DNA, R A or a hybrid thereof.
  • the method comprises a first nucleic acid, and the first nucleic acid is single stranded or double stranded.
  • the method comprises a second nucleic acid, and the second nucleic acid is DNA, RNA or a hybrid thereof.
  • the method comprises a second nucleic acid, and the second nucleic acid is single stranded or double stranded.
  • the method comprises introducing a first nucleic acid, and introducing the first nucleic acid comprises non-viral transfection, biolistics, chemical transfection, electroporation, nucleofection, heat-shock transfection, lipofection, microinjection, or viral transfection.
  • the method comprises viral transduction, and the viral transduction comprises an adeno-associated virus.
  • the method comprises at least one DNA sensing pathway comprising at least one DNA sensing protein
  • the at least one DNA sensing protein is selected from the group consisting of three prime repair exonuclease 1 (TREX1), DEAD-box helicase 41 (DDX41), DNA-dependent activator of IFN-regulatory factor (DAI), Z-DNA -binding protein 1 (ZBP1), interferon gamma inducible protein 16 (IFI16), leucine rich repeat (In FLU) interacting protein 1 (LRRFIP1), DEAH- box helicase 9 (DHX9), DEAH-box helicase 36 (DHX36), Lupus Ku autoantigen protein p70 (Ku70), X-ray repair complementing defective repair in Chinese hamster cells 6 (XRCC6), stimulator of interferon gene (STING), transmembrane protein 173 (TMEM173), tripartite motif containing 32 (TRIM32), tripartite motif containing 56 (TRIM56), ⁇
  • the method comprises disruption of at least one DNA sensing pathway, and the disruption of the at least one DNA sensing pathway comprises at least partial inhibition of at least one DNA sensing protein by the anti-DNA sensing protein. In some embodiments, the method comprises disruption of at least one DNA sensing pathway, and the disruption of the at least one DNA sensing pathway comprises activation of at least one DNA sensing protein by the anti-DNA sensing protein. In some embodiments, the method comprises at least one anti-DNA sensing protein, and the at least one anti-DNA sensing protein is selected from the group consisting of c-FLiP, HCMV pUL83, DENV
  • the method comprises at least one endogenous immunological checkpoint gene, and the at least one endogenous immunological checkpoint gene is PD-1. In some embodiments, the method comprises at least one endogenous immunological checkpoint gene, and the at least one endogenous
  • immunological checkpoint gene is selected from the group consisting of adenosine A2a receptor
  • ADORA CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), indoleamine 2,3-dioxygenase 1 (IDO l), killer cell immunoglobulin- like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte-activation gene 3
  • LAG3 hepatitis A virus cellular receptor 2
  • HAVCR2 hepatitis A virus cellular receptor 2
  • VISTA T-cell activation
  • CD244 natural killer cell receptor 2B4
  • CISH cytokine inducible SH2- containing protein
  • HPRT hypoxanthine phosphoribosyltransferase 1
  • AAVS SITE E.G. AAVS 1, AAVS2, ETC.
  • C-C motif chemokine receptor 5
  • CD 160 CD 160
  • T-cell immunoreceptor with Ig and ITIM domains TAGIT
  • CD96 CD96
  • CTAM cytotoxic and regulatory T-cell molecule
  • LAIRl leukocyte associated immunoglobulin like receptor l(LAIRl)
  • SIGLEC7 sialic acid binding Ig like lectin 9
  • SIGLEC9 tumor necrosis factor receptor superfamily member 10b
  • TNFRSF10A tumor necrosis factor receptor superfamily member 10a
  • caspase 8 caspase 8
  • caspase 10 caspase 10
  • caspase 3 caspase 6
  • caspase 7 caspase 7
  • FADD Fas associated via death domain
  • FAS Fas cell surface death receptor
  • TGFBRII transforming growth factor beta receptor II
  • TGFBR1 SMAD family member 2
  • SMAD3 SMAD family member 3
  • SMAD family member 4 SKI proto-oncogene
  • SKI SKI-like proto-oncogene
  • TGFB induced factor homeobox l(TGIFl) programmed cell death 1
  • PD-1 cytotoxic T-lymphocyte-associated protein 4
  • CTL4 cytotoxic T-lymphocyte-associated protein 4
  • ILIORA interleukin 10 receptor subunit alpha
  • ILIORB interleukin 10 receptor subunit beta
  • HMOX2 interleukin 6 receptor
  • IL6ST interleukin 6 signal transducer
  • CSK c-src tyrosine kinase
  • glycosphingolipid microdomains 1(PAG1) signaling threshold regulating transmembrane adaptor
  • PHD1, PHD2, PHD3 family of proteins, or guanylate cyclase 1, soluble, beta 3(GUCY1B3), T-cell receptor alpha locus (TRA), T cell receptor beta locus (TRB), egl-9 family hypoxia-inducible factor 1
  • EGLN1 egl-9 family hypoxia-inducible factor 1
  • EGLN2 egl-9 family hypoxia-inducible factor 2
  • EGLN3 egl-9 family hypoxia-inducible factor 3
  • the method comprises at least one endogenous
  • the method comprises a double strand break, and creating the double strand break comprises CRISPR.
  • the method comprises a double strand break, and creating the double strand break comprises CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega-TAL.
  • the method comprises a double strand break, and the double strand break is repaired by insertion of the second transgene encoding an engineered TCR.
  • the method comprises a second nucleic acid, and the second nucleic acid comprises recombination arms, and wherein the second transgene encoding an engineered TCR is flanked by the recombination arms.
  • the method comprises recombination arms, and the recombination arms are at least in part complementary to at least a portion of the at least one endogenous immunological checkpoint gene.
  • an increase in isogenicity between the recombination arms and the at least one endogenous immunological checkpoint gene corresponds to an increase in efficiency of the insertion of the second transgene.
  • the method comprises insertion of the second transgene, and an efficiency of the insertion of the second transgene is measured using fluorescence-activated cell sorting.
  • the method comprises introducing a second nucleic acid, and introducing the second nucleic acid comprises non-viral transfection, biolistics, chemical transfection, electroporation, nucleofection, heat-shock transfection, lipofection, microinjection, or viral transfection.
  • the method comprises insertion of a second transgene, and the insertion of the second transgene encoding an engineered TCR comprises homology directed repair (HDR).
  • HDR homology directed repair
  • the method comprises insertions of a second transgene, and the insertion of the second transgene is assisted by a homologous recombination (HR) enhancer.
  • the method comprises an enhancer, and the enhancer is derived from a viral protein.
  • the method comprises an HR enhancer, and the HR enhancer is selected from the group consisting of E4orf6, E lb55K, Elb55K-H354, Elb55K-H373A, Scr7, L755507, or any combination thereof.
  • the method comprises an HR enhancer, and the HR enhancer is a chemical inhibitor.
  • the methods comprise an HR enhancer, and the HR enhancer inhibits Ligase IV.
  • the method comprises a reduction in cytotoxicity, and the cytotoxicity comprises at least one of DNA cleavage, cell death, apoptosis, nuclear condensation, cell lysis, necrosis, altered cell motility, altered cell stiffness, altered cytoplasmic protein expression, altered membrane protein expression, swelling, loss of membrane integrity, cessation of metabolic activity, hypoactive metabolism, hyperactive metabolism, increased reactive oxygen species, cytoplasmic shrinkage, or any combination thereof.
  • the method comprises measuring viability, and the viability is measured using at least one of fluorescence-activated cell sorting, trypan blue exclusion, CD4+ cell-surface markers, CD8+ cell- surface markers, telomere length, or any combination thereof.
  • the method comprises a subject, and the subject is a human subject.
  • the present disclosure provides methods of making a therapeutically effective composition comprising one or more cells.
  • the method comprises measuring a viability of the one or more cells post gene editing.
  • the method comprises gene editing, and the gene editing comprises introducing into the one or more cells a first nucleic acid.
  • the method comprises a first nucleic acid, and the first nucleic acid comprises a first transgene encoding at least one anti-DNA sensing protein.
  • the method comprises at least one DNA sensing pathway, and the at least one DNA sensing pathway is disrupted within the one or more cells by the at least one anti-DNA sensing protein.
  • the method comprises gene editing, and the gene editing comprises introducing into the one or more cells a second nucleic acid.
  • the method comprises a second nucleic acid, and the second nucleic acid comprises a second transgene encoding an engineered T-cell receptor (TCR.
  • TCR engineered T-cell receptor
  • the method comprises at least one endogenous immunological checkpoint gene, and the at least one endogenous immunological checkpoint gene is disrupted within the one or more cells by an insertion of the second transgene.
  • the method comprises disruption of at least one DNA sensing pathway, and the disruption of the at least one DNA sensing pathway reduces cytotoxicity induced by the second transgene, thereby maintaining or increasing viability of the one or more cells.
  • the method comprises measuring an efficiency of the gene editing of the one or more cells. In some aspects, the method comprises calculating an amount of the one or more cells necessary to effect a therapeutic response when administered to a subject. In some embodiments, the method comprises calculating an amount of cells necessary to effect a therapeutic response, and calculating the amount comprises the measured viability and the measured efficiency.
  • the method comprises contacting the calculated amount of the one or more cells of with at least one excipient.
  • the method comprises measuring the viability, and measuring the viability comprises at least one of fluorescence-activated cell sorting, trypan blue exclusion, CD4+ cell-surface markers, CD 8+ cell-surface markers, telomere length, or any combination thereof.
  • the method comprises one or more cells, and the one or more cells are immune cells.
  • the method comprises one or more cells, and the one or more cells are T cells, naive T cells, CD4+ cells, CD8+ cells, stem cells, induced pluripotent stem cells, progenitor cells, hematopoetic cells, primary cells or any combination thereof.
  • the method comprises a first nucleic acid, and the first nucleic acid is DNA, RNA or a hybrid thereof. In some embodiments, the method comprises a first nucleic acid, and the first nucleic acid is single stranded or double stranded. In some embodiments, the method comprises a second nucleic acid, and the second nucleic acid is DNA, RNA or a hybrid thereof. In some embodiments, the method comprises a second nucleic acid, and the second nucleic acid is single stranded or double stranded.
  • the method comprises introducing a first nucleic acid, and introducing the first nucleic acid comprises non-viral transfection, biolistics, chemical transfection, electroporation, nucleofection, heat-shock transfection, lipofection, microinjection, or viral transfection.
  • the method comprises viral transduction, and the viral transduction comprises an adeno-associated virus.
  • the method comprises at least one DNA sensing pathway comprising at least one DNA sensing protein, and the at least one
  • DNA sensing protein is selected from the group consisting of three prime repair exonuclease 1
  • DAI Z-DNA-binding protein 1
  • ZBP1 Z-DNA-binding protein 1
  • IFI16 interferon gamma inducible protein 16
  • IFI16 interferon gamma inducible protein 16
  • LRRFIPl leucine rich repeat (In FLU) interacting protein 1
  • DEAH-box helicase 9 DEAH-box helicase 9
  • DHX36 Lupus Ku autoantigen protein p70
  • XRCC6 X-ray repair complementing defective repair in Chinese hamster cells 6
  • STING stimulator of interferon gene
  • TMEM173 tripartite motif containing 32
  • TNM56 tripartite motif containing 56
  • CNNB 1 myeloid differentiation primary response 88 (MyD88), absent in melanoma 2
  • AIM2 apoptosis-associated speck-like protein containing a CARD
  • ASC apoptosis-associated speck-like protein containing a CARD
  • pro-caspase-1 pro-caspase-1
  • CASP1 caspase-1
  • pro-interleukin 1 beta pro-IL- ⁇
  • pro-interleukin 18 pro-IL-18
  • interleukin 1 beta IL- ⁇
  • interleukin 18 IL-18
  • interferon regulatory factor 1 IRF1
  • IRF3 interferon regulatory Factor 3
  • IRF7 interferon regulatory factor 7
  • ISRE7 interferon-stimulated response element 7
  • ISREl/7 nuclear factor kappa B
  • NF-KB NF-KB
  • RNA polymerase III RNA Pol III
  • MDA-N melanoma differentiation-associated protein 5
  • LGP2 Laboratory of Genetics and Physiology 2
  • RGP2 retinoic acid-inducible gene 1
  • IPS-1 mitochondrial antiviral-signaling protein
  • TRAF3 TNF receptor associated factor 3
  • TANK TRAF family member associated NFKB activator
  • NAP1 nucleosome assembly protein 1
  • TNF-a tumor necrosis factor alpha
  • IFN l interferon lamba-1
  • the method comprises disruption of at least one DNA sensing pathway, and the disruption of the at least one DNA sensing pathway comprises at least partial inhibition of at least one DNA sensing protein by the anti-DNA sensing protein. In some embodiments, the method comprises disruption of at least one DNA sensing pathway, and the disruption of the at least one DNA sensing pathway comprises activation of at least one DNA sensing protein by the anti-DNA sensing protein. In some embodiments, the method comprises at least one anti-DNA sensing protein, and the at least one anti-DNA sensing protein is selected from the group consisting of c-FLiP, HCMV pUL83, DENV NS2B-NS3, HPV18 E7, hAd5 E1A, HSV1 ICPO,
  • the method comprises at least one endogenous immunological checkpoint gene, and the at least one endogenous immunological checkpoint gene is
  • the method comprises at least one endogenous immunological checkpoint gene, and the at least one endogenous immunological checkpoint gene is selected from the group consisting of adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), indoleamine 2,3- dioxygenase 1 (IDO l), killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail,
  • ADORA adenosine A2a receptor
  • VTCN1 V-set domain containing T cell activation inhibitor 1
  • BTLA B and T lymphocyte associated
  • IDO l indoleamine 2,3- dioxygenase 1
  • killer cell immunoglobulin-like receptor three domains, long cytoplasmic tail
  • KIR3DL1 lymphocyte -activation gene 3
  • LAG3 lymphocyte -activation gene 3
  • HAVCR2 V-domain immunoglobulin suppressor of T-cell activation
  • VISTA V-domain immunoglobulin suppressor of T-cell activation
  • CD244 natural killer cell receptor 2B4
  • CISH cytokine inducible SH2 -containing protein
  • phosphoribosyltransferase 1 HPRT
  • AAVS SITE E.G. AAVS1, AAVS2, ETC.
  • CCR5 chemokine (C-C motif) receptor 5 (gene/pseudogene)
  • CD160 molecule CD 160
  • T-cell immunoreceptor with Ig and ITIM domains TAGIT
  • CD96 molecule CD96
  • CTAM cytotoxic and regulatory T-cell molecule
  • LAIRl leukocyte associated immunoglobulin like receptor l
  • SIGLEC7 sialic acid binding Ig like lectin 7
  • SIGLEC9 SIGLEC9
  • SIGLEC9 tumor necrosis factor receptor superfamily member 10b
  • TNFRSF10A tumor necrosis factor receptor superfamily member 10a
  • caspase 8 CASP8
  • caspase 10 caspase 10
  • CERP3 caspase 6
  • caspase 6 CASP6
  • SMAD2 SMAD family member 3
  • SMAD3 SMAD family member 4
  • SKI proto- oncogene SKI
  • SKI-like proto-oncogene SKIL
  • TGFB induced factor homeobox l TGIFl
  • programmed cell death 1 PD-1
  • CTL4 cytotoxic T-lymphocyte-associated protein 4
  • ILIORA interleukin 10 receptor subunit alpha
  • ILIORB interleukin 10 receptor subunit beta
  • HMOX2 interleukin 6 receptor
  • IL6R interleukin 6 receptor
  • IL6ST interleukin 6 signal transducer
  • CSK c-src tyrosine kinase
  • PAG1 glycosphingolipid microdomains 1
  • SIT1 signaling threshold regulating transmembrane adaptor 1(SIT1), forkhead box P3(FOXP3)
  • PR domain l(PRDMl) basic leucine zipper transcription factor
  • BATF guanylate cyclase 1, soluble, alpha 2(GUCY1A2), guanylate cyclase 1, soluble, alpha 3(GUCY1A3), guanylate cyclase 1, soluble, beta 2(GUCY1B2), prolyl hydroxylase domain (PHD1, PHD2, PHD3) family of proteins, or guanylate cyclase 1, soluble, beta 3(GUCY1B3), T-cell receptor alpha locus (TRA), T cell receptor
  • the method comprises at least one endogenous immunological checkpoint gene, and the at least one endogenous immunological checkpoint gene comprises a double strand break.
  • the method comprises a double strand break, and creating the double strand break comprises CRISPR.
  • the method comprises a double strand break, and creating the double strand break comprises CRISPR, TALEN, transposon-based, ZEN, meganuclease, or
  • the method comprises a double strand break, and the double strand break is repaired by insertion of the second transgene encoding an engineered TCR.
  • the method comprises a second nucleic acid, and the second nucleic acid comprises recombination arms, and wherein the second transgene encoding an engineered TCR is flanked by the recombination arms.
  • the method comprises recombination arms, and the recombination arms are at least in part complementary to at least a portion of the at least one endogenous immunological checkpoint gene.
  • an increase in isogenicity between the recombination arms and the at least one endogenous immunological checkpoint gene corresponds to an increase in efficiency of the insertion of the second transgene.
  • the method comprises insertion of the second transgene, and an efficiency of the gene editing corresponds to the efficiency of the insertion of the second transgene.
  • the method comprises measuring an efficiency of the gene editing, and measuring the efficiency of the gene editing comprises at least one of fluorescence-activated cell sorting, realtime PCR, or digital droplet PCR.
  • the method comprises introducing a second nucleic acid, and introducing the second nucleic acid comprises non-viral transfection, biolistics, chemical transfection, electroporation, nucleofection, heat-shock transfection, lipofection, microinjection, or viral transfection.
  • the method comprises insertion of a second transgene, and the insertion of the second transgene encoding an engineered TCR comprises homology directed repair (HDR).
  • the method comprises insertions of a second transgene, and the insertion of the second transgene is assisted by a homologous recombination (HR) enhancer.
  • the method comprises an enhancer, and the enhancer is derived from a viral protein.
  • the method comprises an HR enhancer, and the HR enhancer is selected from the group consisting of E4orf6, E lb55K, Elb55K-H354, Elb55K-H373A, Scr7, L755507, or any combination thereof.
  • the method comprises an HR enhancer, and the HR enhancer is a chemical inhibitor.
  • the methods comprise an HR enhancer, and the HR enhancer inhibits Ligase IV.
  • the method comprises a reduction in cytotoxicity, and the cytotoxicity comprises at least one of DNA cleavage, cell death, apoptosis, nuclear condensation, cell lysis, necrosis, altered cell motility, altered cell stiffness, altered cytoplasmic protein expression, altered membrane protein expression, swelling, loss of membrane integrity, cessation of metabolic activity, hypoactive metabolism, hyperactive metabolism, increased reactive oxygen species, cytoplasmic shrinkage, or any combination thereof.
  • the method comprises an amount of the one or more cells necessary to effect a therapeutic response, and the amount of the one or more cells necessary to effect a therapeutic response when administered to a subject comprises about 5 ⁇ 10 ⁇ 10 cells.
  • the method comprises an amount of the one or more cells necessary to effect a therapeutic response, and the amount of the one or more cells necessary to effect a therapeutic response when administered to a subject comprises at least about 5x10 ⁇ 7 cells.
  • the method comprises one or more cells, and the one or more cells are viable cells.
  • the method comprises a second transgene, and the second transgene is inserted into the at least one endogenous immunological checkpoint gene in the one or more cells.
  • the method comprises a subject, and the subject is a human subject.
  • the method comprises a therapeutic response, and the therapeutic response comprises preventing, reducing, or eliminating cancer in the subject.
  • the method comprises cancer, and the cancer is bladder cancer, bone cancer, a brain tumor, breast cancer, esophageal cancer, gastrointestinal cancer, hematopoietic malignancy, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, prostate cancer, sarcoma, stomach cancer, or thyroid cancer.
  • the method comprises at least one excipient, and the at least one excipient is selected from the group consisting of acetate, acid, alcohol, alginate, ammonium, cell media, cellulose, chitosan, collagen, dextran, dextrose, ester, ethanol, gelatin, glucose, glycerol, lactose, mannitol, mannose, mercurial compounds, mineral oil, phenol, phosphate, polyacrylic acid, polyethylene glycol (PEG), Ringer's solution, saline, sorbitol, starch, sucrose, vegetable oil, water, white petroleum or a combination thereof.
  • the method comprises administering to a subject an amount of engineered cells necessary to effect a therapeutic response in the subject.
  • FIG. 1 is an overview of some of the methods disclosed herein.
  • FIG. 2 shows some exemplary transposon constructs for TCR transgene integration and TCR
  • FIG. 3 demonstrates the in vitro transcription of mRNA and its use as a template to generate
  • homologous recombination (HR) substrate in any type of cell (e.g., primary cells, cell lines, etc.).
  • HR homologous recombination
  • mRNAs encoding both the sense and anti- sense strand of the viral vector can be used to improve yield.
  • FIG. 4 demonstrates the structures of four plasmids, including Cas9 nuclease plasmid, HPRT gRNA plasmid, Amaxa EGFPmax plasmid and HPRT target vector.
  • FIG. 5 shows an exemplary HPRT target vector with targeting arms of 0.5 kb.
  • FIG. 6 demonstrates three potential TCR transgene knock-in designs targeting an exemplary gene (e.g., HPRT gene).
  • TCR TCR transgene
  • Promoter TCR transgene
  • SA in-frame transcription TCR transgene transcribed by endogenous promoter (indicated by the arrow) via splicing
  • Fusion in frame translation TCR transgene transcribed by endogenous promoter via in frame translation.
  • All three exemplary designs can knock- out the gene function. For example, when a HPRT gene or a PD-1 gene is knocked out by insertion of a TCR transgene, a 6-thiogaunine selection can be used as the selection assay.
  • FIG. 7 demonstrates that Cas9+gRNA+Target plasmids co-transfection had good transfection
  • FIG. 8 demonstrates the results of the EGFP FACS analysis of CD3+ T cells.
  • FIG. 9 shows two types of T cell receptors.
  • FIG. 10 shows successful T cell transfection efficiency using two platforms.
  • FIG. 11 shows efficient transfection as T cell number is scaled up, e.g., as T cell number increases.
  • FIG. 12 shows % gene modification occurring by CRISPR gRNAs at potential target sites.
  • FIG. 13 demonstrates CRISPR-induced DSBs in stimulated T cells.
  • FIG. 14 shows optimization of RNA delivery.
  • FIG. 15 demonstrates double strand breaks at target sites. The gene targeting was successful in
  • immune checkpoint genes PD-1, CCR5, and CTLA4 were used to validate the system.
  • FIG. 16 shows a representation of TCR integration at CCR5.
  • the 3kb TCR expression transgene can be inserted into a similar vector with recombination arms to a different gene in order to target other genes of interest using homologous recombination. Analysis by PCR using primers outside of the recombination arms can demonstrate successful TCR integration at a gene.
  • FIG. 17 depicts TCR integration at the CCR5 gene in stimulated T cells. Positive PCR results
  • FIG. 18 shows T death in response to plasmid DNA transfection.
  • FIG.19 is schematic of the innate immune sensing pathway of cytosolic DNA present in different types of cells, including but not limited to T cells.
  • T cells express both pathways for detecting foreign DNA. The cellular toxicity can result from activation of these pathways during genome engineering.
  • FIG. 20 demonstrates that the inhibitors of FIG. 19 block apoptosis and pyropoptosis.
  • FIG. 21 shows a schematic of representative plasmid modifications.
  • a standard plasmid contains bacterial methylation that can trigger an innate immune sensing system. Removing bacterial methylation can reduce toxicity caused by a standard plasmid. Bacterial methylation can also be removed and mammalian methylation added so that the vector looks like "self-DNA.” A modification can also include the use of a synthetic single stranded DNA.
  • FIG. 22 shows a representative functional engineered TCR antigen receptor.
  • This engineered TCR is highly reactive against MART-1 expressing melanoma tumor cell lines.
  • the TCR a and ⁇ chains are linked with a furin cleavage site, followed by a 2A ribosomal skip peptide.
  • FIG. 23 A and FIG. 23 B show PD-1, CTLA-4, PD-1 and CTLA-2, or CCR5, PD-1, and CTLA-4 expression on day 6 post transfection with guide RNAs.
  • A. shows percent inhibitory receptor expression.
  • B. shows normalized inhibitory receptor expression to a control guide RNA.
  • FIG. 24 A and FIG. 24 B shows CTLA -4 expression in primary human T cells after electroporation with CRISPR and CTLA -4 specific guideRNAs, guides #2 and #3, as compared to unstained and a no guide control.
  • B. shows PD-1 expression in primary human T cells after electroporation with CRISPR and PD-1 specific guideRNAs, guides #2 and #6, as compared to unstained and a no guide control.
  • FIG. 25 shows FACs results of CTLA -4 and PD-1 expression in primary human T cells after
  • FIG. 26 A and FIG. 26 B show percent double knock out in primary human T cells post treatment with CRISPR.
  • A. shows percent CTLA-4 knock out in T cells treated with CTLA-4 guides #2, #3, #2 and #3, PD-1 guide #2 and CTLA-4 guide #2, PD-1 guide #6 and CTLA-4 guide #3, as compared to Zap only, Cas9 only, and an all guideRNA control.
  • B. shows percent PD-1 knock out in T cells treated with PD-1 guide#2, PD-1 guide #6, PD-1 guides #2 and #6, PD-1 guide #2 and CTLA-4 guide #2, PD-1 guide #6 and CTLA-4 guide #3, as compared to Zap only, Cas9 only, and an all guideRNA control.
  • FIG. 27 shows T cell viability post electroporation with CRISPR and guide RNAs specific to CTLA- 4, PD-1, or combinations.
  • FIG. 28 results of a CEL-I assay showing cutting by PD-1 guide RNAs #2, #6, #2 and #6, under conditions where only PD-1 guide RNA is introduced, PD-1 and CTLA-4 guide RNAs are introduced or CCR5, PD-1, and CLTA-4 guide RNAs, Zap only, or gRNA only controls.
  • FIG. 29 results of a CEL-I assay showing cutting by CTLA-4 guide RNAs #2, #3, #2 and #3, under conditions where only CLTA-4 guide RNA is introduced, PD-1 and CTLA-4 guide RNAs are introduced or CCR5, PD-1, and CLTA-4 guide RNAs, Zap only, or gRNA only controls.
  • FIG. 30 results of a CEL-I assay showing cutting by CCR5 guide RNA #2 in conditions where CCR5 guide RNA is introduced, CCR5 guide RNA, PD-1 guide RNA, or CTLA-4 guide RNA, as compared to Zap only, Cas 9 only, or guide RNA only controls.
  • FIG. 31 shows knockout of TCR alpha, as measured by CD3 FACs expression, in primary human T cells utilizing optimized CRISPR guideRNAs with 2' O-Methyl RNA modification at 5 micrograms and 10 micrograms.
  • FIG. 32 depicts a method of measuring T cell viability and phenotype post treatment with CRISPR and guide RNAs to CTLA-4. Phenotype was measured by quantifying the frequency of treated cells exhibiting a normal FSC/SSC profile normalized to frequency of electroporation alone control. Viability was also measured by exclusion of viability dye by cells within the FSC/SSC gated population. T cell phenotype is measured by CD3 and CD62L.
  • FIG. 33 shows method of measuring T cell viability and phenotype post treatment with CRISPR and guide RNAs to PD-1, and PD-1 and CTLA-4.
  • Phenotype was measured by quantifying the frequency of treated cells exhibiting a normal FSC/SSC profile normalized to frequency of electroporation alone control. Viability was also measured by exclusion of viability dye by cells within the FSC/SSC gated population.
  • T cell phenotype is measured by CD3 and CD62L.
  • FIG. 34 shows results of a T7E1 assay to detect CRISPR gene editing on day 4 post transfection with PD-1 or CTKA-4 guide RNA of primary human T cells and Jurkat control.
  • N is a no T7E1 nuclease control.
  • FIG. 35 shows results of a tracking of indels by decomposition (TIDE) analysis. Percent gene editing efficiency as shows to PD-1 and CTLA-4 guide RNAs.
  • FIG. 36 shows results of a tracking of indels by decomposition (TIDE) analysis for single guide transfections. Percent of sequences with either deletions or insertions are shown for primary human T cells transfected with PD-1 or CTLA-1 guide RNAs and CRISPR.
  • TIDE indels by decomposition
  • FIG. 37 shows PD-1 sequence deletion with dual targeting.
  • FIG. 38 shows sequencing results of PCR products of PD-1 sequence deletion with dual targeting.
  • Samples 6 and 14 are shown with a fusion of the two gRNA sequences with the intervening 135bp excised.
  • FIG. 39 shows dual targeting sequence deletion of CTLA-4. Deletion between the two guide RNA sequences is also present in the sequencing of dual guide targeted CTLA-4 (samples 9 and 14).
  • T7E1 Assay confirms the deletion by PCR.
  • FIG. 40 A and FIG. 40 B show A. viability of human T cells on day 6 post CRISPR transfection. B.
  • FIG. 41 shows FACs analysis of CTLA-4 expression in stained human T cells transfected with anti-
  • CTLA-4 CRISPR guide RNAs.
  • PE is anti-human CD152 (CTLA-4).
  • FIG. 42 A and FIG. 42 B show CTLA-4 FACs analysis of CTLA-4 positive human T cells post transfection with anti-CTLA-4 guide RNAs and CRISPR.
  • B. shows CTLA-4 knock out efficiency relative to a pulsed control in human T cells post transfection with anti-CTLA-4 guide RNAs and
  • FIG. 43 shows minicircle DNA containing an engineered TCR.
  • FIG. 44 depicts modified sgRNA for CISH, PD-1, CTLA4 and AAVS 1.
  • FIG. 45 Depicts FACs results of PD-1 KO on day 14 post transfection with CRISPR and anti-PD-1 guide RNAs.
  • PerCP-Cy5.5 is mouse anti-human CD279 (PD-1).
  • FIG. 46 A and FIG. 46 B A shows percent PD-1 expression post transfection with an anti-PD-1 CRISPR system.
  • B shows percent PD-1 knock out efficiency as compared to Cas9 only control.
  • FIG. 47 shows FACs analysis of the FSC/SSC subset of human T cells transfected with CRISPR system with anti-PD-1 guide #2, anti-PD-1 guide #6, anti-PD l guides #2 and #6, or anti-PD-1 guides #2 and #6 and anti-CTLA-4 guides #2 and #3.
  • FIG. 48 shows FACs analysis of human T cells on day 6 post transfection with CRISPR and anti- CTLA-4 guide RNAs.
  • PE is mouse anti-human CD152 (CTLA-4).
  • FIG. 49 shows FACs analysis of human T cells and control Jurkat cells on day 1 post transfection with CRISPR and anti-PD-1 and anti-CTLA-4 guide RNAs. Viability and transfection efficiency of human T cells is shown as compared to transfected Jurkat cells.
  • FIG. 50 depicts quantification data from a FACs analysis of CTLA-4 stained human T cells
  • FIG. 51 shows FACs analysis of PD-1 stained human T cells transfected with CRISPR and anti-PD-1 guide RNAs. Day 14 post transfection data is shown of PD-1 expression (anti-human CD279 PerCP-
  • FIG. 52 shows percent PD-1 expression and percent knock out of PD-1 compared to Cas9 only
  • FIG. 53 shows day 14 cell count and viability of transfected human T cells with CRISPR, anti- CTLA-4, and anti-PD-1 guide RNAs.
  • FIG. 54 shows FACs data for human T cells on day 14 post electroporation with CRISPR, and anti- PD-1 guide #2 alone, anti-PD-1 guide #2 and #6, or anti-CTLA-4 guide #3 alone.
  • the engineered T cells were re-stimulated for 48 hours to assess expression of CTLA-4 and PD-1 and compared to control cells electroporated with no guide RNA.
  • FIG. 55 shows FACs data for human T cells on day 14 post electroporation with CRISPR, and anti- CTLA-4 guide #2 and #3, anti-PD-1 guide #2 and anti-CTLA-4 guide #3, or anti-PD-1 guide #2 and #6, anti-CTLA-4 guide #3 and #2.
  • the engineered T cells were re-stimulated for 48 hours to assess expression of CTLA-4 and PD-1 and compared to control cells electroporated with no guide RNA.
  • FIG. 56 depicts results of a surveyor assay for CRISPR mediated gene -modification of the CISH locus in primary human T cells.
  • FIG. 57 A, FIG. 57 B, and FIG. 57 C A depict a schematic of a T cell receptor (TCR).
  • TCR T cell receptor
  • B. shows a schematic of a chimeric antigen receptor.
  • C. shows a schematic of a B cell receptor (BCR).
  • FIG. 58 Shows that somatic mutational burden varies among tumor type. Tumor-specific neo-antigen generation and presentation is theoretically directly proportional to mutational burden.
  • FIG. 59 shows pseudouridine-5 '-Triphosphate and 5 -Methylcytidine -5 -Triphosphate modifications that can be made to nucleic acid.
  • FIG. 60 shows TIDE and densitometry data comparison for 293T cells transfected with CRISPR and CISH gRNAs 1,3,4,5 or 6.
  • FIG. 61 depicts duplicate experiments of densitometry analysis for 293T cells transfected with
  • FIG. 62 A and FIG. 62 B show duplicate TIDE analysis A. and B. of CISH gRNA 1.
  • FIG. 63 A and FIG. 63 B show duplicate TIDE analysis A. and B. of CISH gRNA 3.
  • FIG. 64 A and FIG. 64 B show duplicate TIDE analysis A. and B. of CISH gRNA 4.
  • FIG. 65 A and FIG. 65 B show duplicate TIDE analysis A. and B. of CISH gRNA 5.
  • FIG. 66 A and FIG. 66 B show duplicate TIDE analysis A. and B. of CISH gRNA 6.
  • FIG. 67 shows a western blot showing loss of CISH protein after CRISPR knock out in primary T cells.
  • FIG. 68 A, FIG. 68 B, and FIG. 68 C depict DNA viability by cell count A. 1 day, B. 2 days, C. 3 days post transfection with single or double -stranded DNA.
  • M13 ss/dsDNA is 7.25 kb.
  • pUC57 is 2.7 kb.
  • GFP plasmid is 6.04 kb.
  • FIG. 69 shows a mechanistic pathway that can be modulated during preparation or post preparation of engineered cells.
  • FIG. 70 A and FIG. 70 B depict cell count post transfection with the CRISPR system (15ug Cas9, lOug gRNA) on A. Day 3 and B. Day 7.
  • Sample 7- plasmid donor (5 micrograms).
  • FIG. 71 A and FIG. 71 B shows Day 4 TIDE analysis of PDl A. gRNA 2 and B. gRNA6 with no donor nucleic acid.
  • FIG. 72 A and FIG. 72 B shows Day 4 TIDE analysis of CTLA4 A. gRNA 2 and B. gRNA3 with no donor nucleic acid.
  • FIG. 73 shows FACs analysis of day 7 TCR beta detection in control cells, cells electroporated with 5 micrograms of donor DNA (minicircle), or cells electroporated with 20 micrograms of donor DNA (minicircle).
  • FIG. 74 shows a summary of day 7 T cells electroporated with the CRISPR system and either no polynucleic acid donor (control), 5 micrograms of polynucleic acid donor (minicircle), or 20 micrograms of polynucleic acid donor (minicircle). A summary of FACs analysis of TCR positive cells is shown.
  • FIG. 75 shows integration of the TCR minicircle in the forward direction into the PDl gRNA#2 cut site.
  • FIG. 76 A and FIG. 76 B shows percentage of live cells at day 4 using a GUIDE-Seq dose test of human T cells transfected with CRISPR and PD-1 or CISH gRNAs with 5' or 3' modifications (or both) at increasing concentrations of a double stranded polynucleic acid donor.
  • B. shows efficiency of integration at the PD-1 or CISH locus of human T cells transfected with CRISPR and PD-1 or CISH specific gRNAs.
  • FIG. 77 shows GoTaq and PhusionFlex analysis of dsDNA integration at the PD-1 or CISH gene sites.
  • FIG. 78 shows day 15 FACs analysis of human T cells transfected with CRISPR and 5 micrograms or
  • FIG. 79 shows a summary of day 15 T cells electroporated with the CRISPR system and either no polynucleic acid donor (control), 5 micrograms of polynucleic acid donor (minicircle), or 20 micrograms of polynucleic acid donor (minicircle). A summary of FACs analysis of TCR positive cells is shown.
  • FIG. 80 depicts digital PCR copy number data copy number relative to RNaseP on Day 4 post
  • FIG. 81 A. and FIG. 81 B. show A. Day 3 T cell viability with increasing dose of minicircle
  • FIG. 82 A. and FIG. 82 B. show A. optimization conditions for Lonza nucleofection of T cell double strand DNA transfection. Cell number vs concentration of a plasmid encoding GFP. B. optimization conditions for Lonza nucleofection of T cells with double strand DNA encoding a GFP protein. Percent transduction is shown vs concentration of GFP plasmid used for transfection.
  • A. depict a pDG6-AAV helper-free packaging plasmid for AAV TCR delivery.
  • B. shows a schematic of a protocol for AAV transient transfection of 293 cells for virus production. Virus will be purified and stored for transduction into primary human T cells.
  • FIG. 84 shows a rAAV donor encoding an exogenous TCR flanked by 900bp homology arms to an endogenous immune checkpoint (CTLA4 and PD 1 are shown as exemplary examples).
  • FIG. 85 shows a genomic integration schematic of a rAAV homologous recombination donor
  • FIG. 86 A, FIG. 86 B, FIG. 86 C, and FIG. 86 D show possible recombination events that may occur using the AAVS1 system.
  • A. shows homology directed repair of double stand breaks at AAVS1 with integration of the transgene.
  • B. shows homology directed repair of one stand of the AAVS1 gene and non-homologous end joining indel of the complementary stand of AAVS1.
  • C. shows nonhomologous end joining insertion of the transgene into the AAVS 1 gene site and non-homologous end joining indel at AAVS1.
  • D. shows nonhomologous idels at both AAVS1 locations with random integration of the transgene into a genomic site.
  • FIG. 87 shows a combined CRISPR and rAAV targeting approach of introducing a transgene
  • FIG. 88 A and FIG 88. B show day 3 data
  • A CRISPR electroporation experiment in which caspase and TBK inhibitors were used during the electroporation of a 7.5 microgram minicircle donor encoding an exogenous TCR. Viability is plotted in comparison to concentration of inhibitor used. B. shows efficiency of electroporation. Percent positive TCR is shown vs. concentration of inhibitor used.
  • FIG. 89 shows FACs data of human T cells electroporated with CRISPR and minicircle DNA (7.5 microgram) encoding an exogenous TCR. Caspase and TBK inhibitors were added during the electroporation.
  • FIG. 90A and FIG. 90B show FACs data of human T cells electroporated with CRISPR and a
  • FIG. 91 shows TCR expression on day 13 post electroporation with CRISPR and a minicircle
  • FIG. 92A and FIG.92B shows a cell death inhibitor study in which human T cells were pre-treated with Brefeldin A and ATM-inhibitors prior to transfection with CRISPR and minicircle DNA encoding for an exogenous TCR.
  • A. shows viability of T cells on day 3 post electroporation.
  • B. shows viability of T cells on day 7 post electroporation.
  • FIG. 93A and FIG. 93B shows a cell death inhibitor study in which human T cells were pre-treated with Brefeldin A and ATM-inhibitors prior to transfection with CRISPR and minicircle DNA encoding for an exogenous TCR.
  • A. shows TCR expression on T cells on day 3 post electroporation.
  • B. shows TCR expression on T cells on day 7 post electroporation.
  • FIG. 94 shows a splice -acceptor GFP reporter assay to rapidly detect integration of an exogenous transgene (e.g. , TCR).
  • an exogenous transgene e.g. , TCR
  • FIG. 95 shows a locus-specific digital PCR assay to rapidly detect integration of an exogenous
  • transgene e.g., TCR
  • FIG. 96 shows recombinant (rAAV) donor constructs encoding for an exogenous TCR using either a PGK promoter or a splice acceptor. Each construct is flanked by 850 base pair homology arms (HA) to the AAVS 1 checkpoint gene.
  • rAAV recombinant
  • FIG. 97 shows the rAAV AAVS l-TCR gene targeting vector.
  • Major features are shown along with their sizes in numbers of nucleotides (bp).
  • ITR internal tandem repeat
  • PGK phosphoglycerate kinase
  • mTCR murine T-cell receptor beta
  • SV40 PolyA Simian virus 40 polyadenylation signal.
  • FIG. 98 shows T cells electroporated with a GFP+ transgene 48 hours post stimulation with modified gRNAs.
  • gRNAs were modified with pseudouridine, 5 'moC, 5'meC, 5 'moU, 5 'hmC+5'moU, m6A, or 5 'moC+5 'meC.
  • FIG. 99 A and FIG 99 B depeict A. viability and B. MFI of GFP expressing cells for T cells
  • gRNAs were modified with pseudouridine, 5 'moC, 5 'meC, 5'moU, 5 'hmC+5 'moU, m6A, or 5 'moC+5 'meC.
  • FIG. 100 A and FIG 100 B show TIDE results of a comparison of a A. modified clean cap Cas9 protein or an B. unmodified Cas9 protein. Genomic integration was measured at the CCR5 locus of T cells electroporated with unmodified Cas9 or clean cap Cas9 at 15 micrograms of Cas9 and 10 micrograms of a chemically modified gRNA.
  • FIG. 101 A and FIG. 101 B show A. viability and B. reverse transcriptase activity for Jurkat cells expressing revese transcriptiase (RT) reporter RNA that were transfected using the Neon Transfection System with RT encoding plasmids and primers (see table for concentrations) and assayed for cell viability and GFP expression on Days 3 post transfection.
  • GFP positive cells represent cells with RT activity.
  • FIG. 102 A and FIG. 102 B show absoluate cell count pre and post stimulation of human TILs. A.
  • FIG. 1 shows a first donor's cell count pre- and post- sitmulation cultured in either RPMI media or ex vivo media.
  • B. shows a second donor's cell count pre- and post- stimulation cultured in RPMI media.
  • FIG. 103 A and FIG 103 B show cellular expansion of human tumor infilatrating lymphocytes (TILs) electroporated with a CRISPR system targeting PD-1 locus or controls cells A. with the addition of autologous feeders or B. without the addition of autologous feeders.
  • TILs tumor infilatrating lymphocytes
  • FIG. 104A and FIG. 104 B show human T cells electroporated with the CRISPR system alone
  • GFP plasmid donor alone (control); donor and CRISPR system; donor, CRISPR, and cFLP protein; donor, CRISPR, and hAd5 El A (El A) protein; or donor, CRISPR, and HPV18 E7 (E7) protein.
  • FACs analysis of GFP was measured at A. 48 hours or B. 8 days post electroporation.
  • the term "about” and its grammatical equivalents in relation to a reference numerical value and its grammatical equivalents as used herein can include a range of values plus or minus 10% from that value.
  • the amount “about 10” includes amounts from 9 to 11.
  • the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.
  • activation and its grammatical equivalents as used herein can refer to a process whereby a cell transitions from a resting state to an active state. This process can comprise a response to an antigen, migration, and/or a phenotypic or genetic change to a functionally active state.
  • activation can refer to the stepwise process of T cell activation.
  • a T cell can require at least two signals to become fully activated. The first signal can occur after engagement of a TCR by the antigen-MHC complex, and the second signal can occur by engagement of co-stimulatory molecules.
  • Anti-CD3 can mimic the first signal and anti-CD28 can mimic the second signal in vitro.
  • adjacent and its grammatical equivalents as used herein can refer to right next to the object of reference.
  • adjacent in the context of a nucleotide sequence can mean without any nucleotides in between.
  • polynucleotide A adjacent to polynucleotide B can mean AB without any nucleotides in between A and B.
  • antigen and its grammatical equivalents as used herein can refer to a molecule that
  • an antigen can stimulate a host's immune system to make a cellular antigen-specific immune response when the antigen is presented, or a humoral antibody response.
  • An antigen can also have the ability to elicit a cellular and/or humoral response by itself or when present in combination with another molecule.
  • a tumor cell antigen can be recognized by a TCR.
  • epitope and its grammatical equivalents as used herein can refer to a part of an antigen that can be recognized by antibodies, B cells, T cells or engineered cells.
  • an epitope can be a cancer epitope that is recognized by a TCR. Multiple epitopes within an antigen can also be recognized. The epitope can also be mutated.
  • autologous and its grammatical equivalents as used herein can refer to as originating from the same being.
  • a sample e.g., cells
  • An autologous process is distinguished from an allogenic process where the donor and the recipient are different subjects.
  • barcoded to refers to a relationship between molecules where a first molecule contains a barcode that can be used to identify a second molecule.
  • cancer and its grammatical equivalents as used herein can refer to a hyperproliferation of cells whose unique trait— loss of normal controls— results in unregulated growth, lack of
  • the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, rectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mastocytoma,
  • cancer neo-antigen or “neo-antigen” or “neo-epitope” and its grammatical equivalents as used herein can refer to antigens that are not encoded in a normal, non-mutated host genome.
  • a "neo- antigen” can in some instances represent either oncogenic viral proteins or abnormal proteins that arise as a consequence of somatic mutations.
  • a neo-antigen can arise by the disruption of cellular mechanisms through the activity of viral proteins.
  • Another example can be an exposure of a carcinogenic compound, which in some cases can lead to a somatic mutation. This somatic mutation can ultimately lead to the formation of a tumor/cancer.
  • cytotoxicity refers to an unintended or undesirable alteration in the normal state of a cell.
  • the normal state of a cell may refer to a state that is manifested or exists prior to the cell's exposure to a cytotoxic composition, agent and/or condition.
  • a cell that is in a normal state is one that is in homeostasis.
  • An unintended or undesirable alteration in the normal state of a cell can be manifested in the form of, for example, cell death (e.g., programmed cell death), a decrease in replicative potential, a decrease in cellular integrity such as membrane integrity, a decrease in metabolic activity, a decrease in developmental capability, or any of the cytotoxic effects disclosed in the present application.
  • reducing cytotoxicity or “reduce cytotoxicity” refers to a reduction in degree
  • the phrase can refer to reducing the degree of cytotoxicity in an individual cell that is exposed to a cytotoxic composition, agent and/or condition, or to reducing the number of cells of a population that exhibit cytotoxicity when the population of cells is exposed to a cytotoxic composition, agent and/or condition.
  • engineered and its grammatical equivalents as used herein can refer to one or more
  • nucleic acid e.g., the nucleic acid within an organism's genome.
  • engineered can refer to alterations, additions, and/or deletion of genes.
  • An engineered cell can also refer to a cell with an added, deleted and/or altered gene.
  • cell or “engineered cell” and their grammatical equivalents as used herein can refer to a cell of human or non-human animal origin.
  • checkpoint gene and its grammatical equivalents as used herein can refer to any gene that is involved in an inhibitory process (e.g., feedback loop) that acts to regulate the amplitude of an immune response, for example, an immune inhibitory feedback loop that mitigates uncontrolled propagation of harmful responses. These responses can include contributing to a molecular shield that protects against collateral tissue damage that might occur during immune responses to infections and/or maintenance of peripheral self-tolerance.
  • inhibitory process e.g., feedback loop
  • These responses can include contributing to a molecular shield that protects against collateral tissue damage that might occur during immune responses to infections and/or maintenance of peripheral self-tolerance.
  • Non-limiting examples of checkpoint genes can include members of the extended CD28 family of receptors and their ligands as well as genes involved in co-inhibitory pathways (e.g., CTLA-4 and PD-1).
  • the term "checkpoint gene” can also refer to an immune checkpoint gene.
  • CRISPR CRISPR system system
  • CRISPR nuclease system CRISPR nuclease system
  • RNA equivalents can include a non-coding RNA molecule (e.g., guide RNA) that binds to DNA and Cas proteins (e.g., Cas9) with nuclease functionality (e.g. , two nuclease domains).
  • Cas proteins e.g., Cas9
  • nuclease functionality e.g. , two nuclease domains.
  • disrupting and its grammatical equivalents as used herein can refer to a process of altering a gene, e.g. , by deletion, insertion, mutation, rearrangement, or any combination thereof.
  • a gene can be disrupted by knockout.
  • Disrupting a gene can be partially reducing or completely suppressing expression of the gene.
  • Disrupting a gene can also cause activation of a different gene, for example, a downstream gene.
  • the term "function" and its grammatical equivalents as used herein can refer to the capability of operating, having, or serving an intended purpose.
  • Functional can comprise any percent from baseline to 100% of normal function.
  • functional can comprise or comprise about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, and/or 100% of normal function.
  • the term functional can mean over or over about 100% of normal function, for example, 125, 150, 175, 200, 250, 300% and/or above normal function.
  • nuclease e.g., a natural-existing nuclease or an artificially engineered nuclease.
  • mutants can include the substitution, deletion, and insertion of one or more nucleotides in a polynucleotide. For example, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or more nucleotides/amino acids in a polynucleotide (cDNA, gene) or a polypeptide sequence can be substituted, deleted, and/or inserted.
  • a mutation can affect the coding sequence of a gene or its regulatory sequence.
  • a mutation can also affect the structure of the genomic sequence or the structure/stability of the encoded mRNA.
  • non-human animal and its grammatical equivalents as used herein can include all animal species other than humans, including non-human mammals, which can be a native animal or a genetically modified non-human animal.
  • non-human mammals which can be a native animal or a genetically modified non-human animal.
  • nucleic acid polynucleotide
  • polynucleic acid polynucleic acid
  • oligonucleotide and their grammatical equivalents can be used interchangeably and can refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For the purposes of the present disclosure, these terms should not to be construed as limiting with respect to length.
  • the terms can also encompass analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g. , phosphorothioate backbones). Modifications of the terms can also encompass demethylation, addition of CpG methylation, removal of bacterial methylation, and/or addition of mammalian methylation.
  • an analogue of a particular nucleotide can have the same base- pairing specificity, i.e. , an analogue of A can base-pair with T.
  • peripheral blood lymphocytes can refer to lymphocytes that circulate in the blood (e.g. , peripheral blood).
  • Peripheral blood lymphocytes can refer to lymphocytes that are not localized to organs.
  • Peripheral blood lymphocytes can comprise T cells, NK cells, B cell, or any combinations thereof.
  • phenotype and its grammatical equivalents as used herein can refer to a composite of an organism's observable characteristics or traits, such as its morphology, development, biochemical or physiological properties, phenology, behavior, and products of behavior. Depending on the context, the term “phenotype” can sometimes refer to a composite of a population's observable characteristics or traits.
  • protospacer and its grammatical equivalents as used herein can refer to a PAM-adjacent nucleic acid sequence capable to hybridizing to a portion of a guide R A, such as the spacer sequence or engineered targeting portion of the guide RNA.
  • a protospacer can be a nucleotide sequence within gene, genome, or chromosome that is targeted by a guide RNA. In the native state, a protospacer is adjacent to a PAM (protospacer adjacent motif). The site of cleavage by an RNA-guided nuclease is within a protospacer sequence.
  • the Cas protein when a guide RNA targets a specific protospacer, the Cas protein will generate a double strand break within the protospacer sequence, thereby cleaving the protospacer.
  • disruption of the protospacer can result though non-homologous end joining (NHEJ) or homology-directed repair (HDR).
  • NHEJ non-homologous end joining
  • HDR homology-directed repair
  • Disruption of the protospacer can result in the deletion of the protospacer.
  • disruption of the protospacer can result in an exogenous nucleic acid sequence being inserted into or replacing the protospacer.
  • recipient and their grammatical equivalents as used herein can refer to a human or non- human animal. The recipient can also be in need thereof.
  • recombination and its grammatical equivalents as used herein can refer to a process of exchange of genetic information between two polynucleic acids.
  • homologous recombination or “HR” can refer to a specialized form of such genetic exchange that can take place, for example, during repair of double-strand breaks. This process can require nucleotide sequence homology, for example, using a donor molecule to template repair of a target molecule (e.g. , a molecule that experienced the double-strand break), and is sometimes known as non- crossover gene conversion or short tract gene conversion.
  • Such transfer can also involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or synthesis- dependent strand annealing, in which the donor can be used to resynthesize genetic information that can become part of the target, and/or related processes.
  • Such specialized HR can often result in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide can be incorporated into the target polynucleotide.
  • the terms "recombination arms” and “homology arms” can be used interchangeably.
  • target vector and “targeting vector” are used interchangeably herein.
  • transgene and its grammatical equivalents as used herein can refer to a gene or genetic material that is transferred into an organism.
  • a transgene can be a stretch or segment of DNA containing a gene that is introduced into an organism. When a transgene is transferred into an organism, the organism is then referred to as a transgenic organism.
  • a transgene can retain its ability to produce R A or polypeptides (e.g., proteins) in a transgenic organism.
  • a transgene can be composed of different nucleic acids, for example RNA or DNA.
  • a transgene may encode for an engineered T cell receptor, for example a TCR transgene.
  • a transgene may comprise a TCR sequence.
  • a transgene can comprise recombination arms.
  • a transgene can comprise engineered sites.
  • T cell and its grammatical equivalents as used herein can refer to a T cell from any origin.
  • a T cell can be a primary T cell, e.g., an autologous T cell, a cell line, etc.
  • the T cell can also be human or non-human.
  • TIL tumor infiltrating lymphocyte and its grammatical equivalents as used herein can refer to a cell isolated from a tumor.
  • a TIL can be a cell that has migrated to a tumor.
  • a TIL can also be a cell that has infiltrated a tumor.
  • a TIL can be any cell found within a tumor.
  • a TIL can be a T cell, B cell, monocyte, natural killer cell, or any combination thereof.
  • a TIL can be a mixed population of cells.
  • a population of TILs can comprise cells of different phenotypes, cells of different degrees of differentiation, cells of different lineages, or any combination thereof.
  • a "therapeutic effect” may occur if there is a change in the condition being treated.
  • the change may be positive or negative.
  • a 'positive effect' may correspond to an increase in the number of activated T-cells in a subject.
  • a 'negative effect' may correspond to a decrease in the amount or size of a tumor in a subject.
  • There is a "change" in the condition being treated if there is at least 10% improvement, preferably at least 25%, more preferably at least 50%, even more preferably at least 75%, and most preferably 100%.
  • the change can be based on improvements in the severity of the treated condition in an individual, or on a difference in the frequency of improved conditions in populations of individuals with and without treatment with the therapeutic compositions with which the compositions of the present invention are administered in combination.
  • a method of the present disclosure may comprise administering to a subject an amount of cells that is "therapeutically effective".
  • the term "therapeutically effective” should be understood to have a definition corresponding to 'having a therapeutic effect' .
  • safe harbor and "immune safe harbor”, and their grammatical equivalents as used herein can refer to a location within a genome that can be used for integrating exogenous nucleic acids wherein the integration does not cause any significant effect on the growth of the host cell by the addition of the nucleic acid alone.
  • Non-limiting examples of safe harbors can include HPRT, AAVS SITE (E.G. AAVS1, AAVS2, ETC.), CCR5, or Rosa26.
  • sequence and its grammatical equivalents as used herein can refer to a nucleotide
  • sequence which can be DNA or RNA; can be linear, circular or branched; and can be either single- stranded or double stranded.
  • a sequence can be mutated.
  • a sequence can be of any length, for example, between 2 and 1,000,000 or more nucleotides in length (or any integer value there between or there above), e.g. , between about 100 and about 10,000 nucleotides or between about 200 and about 500 nucleotides.
  • An intracellular genomic transplant may comprise genetically modifying cells and nucleic acids for therapeutic applications.
  • the compositions and methods described throughout can use a nucleic acid-mediated genetic engineering process for delivering a tumor-specific TCR in a way that improves physiologic and immunologic anti-tumor potency of an engineered cell.
  • Effective adoptive cell transfer-based immunotherapies can be useful to treat cancer (e.g., metastatic cancer) patients.
  • cancer e.g., metastatic cancer
  • PBL peripheral blood lymphocytes
  • TCR T Cell Receptors
  • a Neoantigen can be associated with tumors of high mutational burden, FIG. 58.
  • Figure 1 depicts and example of a method which can identify a cancer-related target sequence, in some cases a Neoantigen, from a sample obtained from a cancer patient using an in vitro assay (e.g. whole-exomic sequencing).
  • the method can further identify a TCR transgene from a first T cell that recognizes the target sequence.
  • the cancer-related target sequence and a TCR transgene can be obtained from samples of the same patient or different patients.
  • the method can effectively and efficiently deliver a nucleic acid comprising a TCR transgene across membrane of a second T cell.
  • the first and second T cells can be obtained from the same patient. In other instances, the first and second T cells can be obtained from different patients.
  • the first and second T cells can be obtained from different patients.
  • the method can safely and efficiently integrate a TCR transgene into the genome of a T cell using a non-viral integration system (e.g. , CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega-TAL) to generate an engineered T cell and thus, a TCR transgene can be reliably expressed in the engineered T cell.
  • a non-viral integration system e.g. , CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega-TAL
  • the engineered T cell can be grown and expanded in a condition that maintains its immunologic and anti-tumor potency and can further be administered into a patient for cancer treatment.
  • the engineered cell can also be grown and expanded in conditions that can improve its performance once administered to a patient.
  • the engineered cell can be selected.
  • a source of cells can be obtained from a subject through a variety of non-limiting methods.
  • Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • any T cell lines can be used.
  • the cell can be derived from a healthy donor, from a patient diagnosed with cancer, or from a patient diagnosed with an infection.
  • the cell can be part of a mixed population of cells which present different phenotypic characteristics.
  • a cell line can also be obtained from a transformed T- cell according to the method previously described.
  • a cell can also be obtained from a cell therapy bank. Modified cells resistant to an immunosuppressive treatment can be obtained.
  • a desirable cell population can also be selected prior to modification.
  • An engineered cell population can also be selected after modification.
  • the engineered cell can be used in autologous transplantation.
  • the engineered cell can be used in autologous transplantation.
  • engineered cell can be used in allogeneic transplantation.
  • the engineered cell can be administered to the same patient whose sample was used to identify the cancer-related target sequence and/or a TCR transgene.
  • the engineered cell can be administered to a patient different from the patient whose sample was used to identify the cancer-related target sequence and/or a TCR transgene.
  • One or more homologous recombination enhancers can be introduced with cells of the invention. Enhancers can facilitate homology directed repair of a double strand break. Enhancers can facilitate integration of a TCR into a cell of the invention. An enhancer can block non -homologous end joining (NHEJ) so that homology directed repair of a double strand break occurs preferentially.
  • NHEJ non -homologous end joining
  • a modifying compound can also be utilized to reduce toxicity of exogenous polynucleic acids of the invention.
  • a modifier compound can act on Caspase-1, TBK1, IRF3, STING, DDX41, DNA-PK, DAI, IFI16, MRE11, cGAS, 2'3'-cGAMP, TREX1, AIM2, ASC, or any combination thereof.
  • a modifier can be a TBK1 modifier.
  • a modifier can be a caspcase-1 modifier.
  • a modifier compound can also act on the innate signaling system, thus, it can be an innate signaling modifier.
  • exogenous nucleic acids can be toxic to cells.
  • a method that inhibits an innate immune sensing response of cells can improve cell viability of engineered cellular products.
  • a modifying compound can be brefeldin A and or an inhibitor of an ATM pathway, FIG. 92A, FIG.92B, FIG. 93A and FIG. 93B
  • a modifying compound can be introduced to a cell before the addition of a polynucleic acid.
  • modifying compound can be introduced concurrently with a polynucleic acid.
  • a modifying compound can be comprised within a polynucleic acid.
  • cytokines can be introduced with cells of the invention. Cytokines can be utilized to boost cytotoxic T lymphocytes (including adoptively transferred tumor-specific cytotoxic T lymphocytes) to expand within a tumor microenvironment. In some cases, IL-2 can be used to facilitate expansion of the cells described herein. Cytokines such as IL-15 can also be employed. Other relevant cytokines in the field of immunotherapy can also be utilized, such as IL-2, IL-7, IL-12, IL-15, IL-21, or any combination thereof. In some cases, IL-2, IL-7, and IL-15 are used to culture cells of the invention.
  • Cytotoxicity may generally refer to the quality of a composition, agent, and/or condition (e.g.,
  • cytotoxicity or the effects of a substance being cytotoxic to a cell, can comprise DNA cleavage, cell death, autophagy, apoptosis, nuclear condensation, cell lysis, necrosis, altered cell motility, altered cell stiffness, altered cytoplasmic protein expression, altered membrane protein expression, undesired cell differentiation, swelling, loss of membrane integrity, cessation of metabolic activity, hypoactive metabolism, hyperactive metabolism, increased reactive oxygen species, cytoplasmic shrinkage, production of proinflammatory cytokines (e.g., as a product of a DNA sensing pathway) or any combination thereof.
  • proinflammatory cytokines e.g., as a product of a DNA sensing pathway
  • Non-limiting examples of pro-inflammatory cytokines include interleukin 6 (IL-6), interferon alpha (IFNa), interferon beta ( ⁇ ), C-C motif ligand 4 (CCL4), C-C motif ligand 5 (CCL5), C-X-C motif ligand 10 (CXCL10), interleukin 1 beta (IL- ⁇ ), IL-18 and IL-33.
  • cytotoxicity may be affected by introduction of a polynucleic acid, such as a transgene or TCR. Incorporation of an exogenous TCR into a cell may
  • a change in cytotoxicity can be measured in any of a number of ways known in the art. In one
  • a change in cytotoxicity can be assessed based on a degree and/or frequency of occurrence of cytotoxicity-associated effects, such as cell death or undesired cell differentiation.
  • reduction in cytotoxicity is assessed by measuring amount of cellular toxicity using assays known in the art, which include standard laboratory techniques such as dye exclusion, detection of morphologic characteristics associated with cell viability, injury and/or death, and measurement of enzyme and/or metabolic activities associated with the cell type of interest.
  • the T cells of the invention can be expanded by contact with a surface having attached thereto an agent that can stimulate a CD3 TCR complex associated signal and a ligand that can stimulate a co-stimulatory molecule on the surface of the T cells.
  • T cell populations can be stimulated in vitro such as by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) sometimes in conjunction with a calcium ionophore.
  • a ligand that binds the accessory molecule can be used for co-stimulation of an accessory molecule on the surface of the T cells.
  • a population of T cells can be contacted with an anti-CD3 antibody and an anti- CD28 antibody, under conditions that can stimulate proliferation of the T cells.
  • 4-1BB can be used to stimulate cells.
  • cells can be stimulated with 4-1BB and IL-21 or another cytokine.
  • CD28 antibody can be used.
  • the agents providing a signal may be in solution or coupled to a surface.
  • the ratio of particles to cells may depend on particle size relative to the target cell.
  • the cells such as T cells, can be combined with agent-coated beads, where the beads and the cells can be subsequently separated, and optionally cultured. Each bead can be coated with either anti-CD3 antibody or an anti-CD28 antibody, or in some cases, a combination of the two.
  • the agent-coated beads and cells prior to culture, are not separated but are cultured together.
  • Cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 can be attached (3x28 beads) to contact the T cells.
  • the cells and beads are combined in a buffer, for example, phosphate buffered saline (PBS) (e.g., without divalent cations such as, calcium and magnesium). Any cell concentration may be used.
  • PBS phosphate buffered saline
  • Any cell concentration may be used.
  • the mixture may be cultured for or for about several hours (e.g., about 3 hours) to or to about 14 days or any hourly integer value in between. In another embodiment, the mixture may be cultured for or for about 21 days or for up to or for up to about 21 days.
  • Conditions appropriate for T cell culture can include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g , IL-4, IL-7, GM-CSF, IL-10, IL-21, IL-15, TGF beta, and TNF alpha or any other additives for the growth of cells.
  • Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl- cysteine and 2-mercaptoethanol.
  • Media can include RPMI 1640, Al M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 , and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
  • Antibiotics e.g., penicillin and streptomycin, can be included only in experimental cultures, possibly not in cultures of cells that are to be infused into a subject.
  • the target cells can be maintained under conditions necessary to support growth; for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% C0 2 ).
  • an appropriate temperature e.g., 37° C
  • atmosphere e.g., air plus 5% C0 2
  • T cells that have been exposed to varied stimulation times may exhibit different characteristics.
  • a soluble monospecific tetrameric antibody against human CD3, CD28, CD2, or any combination thereof may be used.
  • cells to undergo genomic transplant can be activated or expanded by co-culturing with tissue or cells.
  • a cell can be an antigen presenting cell.
  • An artificial antigen presenting cells (aAPCs) can express ligands for T cell receptor and costimulatory molecules and can activate and expand T cells for transfer, while improving their potency and function in some cases.
  • An aAPC can be engineered to express any gene for T cell activation.
  • An aAPC can be engineered to express any gene for T cell expansion.
  • An aAPC can be a bead, a cell, a protein, an antibody, a cytokine, or any combination.
  • An aAPC can deliver signals to a cell population that may undergo genomic transplant.
  • an aAPC can deliver a signal 1, signal, 2, signal 3 or any combination.
  • a signal 1 can be an antigen recognition signal.
  • signal 1 can be ligation of a TCR by a peptide-MHC complex or binding of agonistic antibodies directed towards CD3 that can lead to activation of the CD3 signal-transduction complex.
  • Signal 2 can be a co-stimulatory signal.
  • a co- stimulatory signal can be anti-CD28, inducible co-stimulator (ICOS), CD27, and 4- IBB (CD 137), which bind to ICOS-L, CD70, and 4-1BBL, respectively.
  • Signal 3 can be a cytokine signal.
  • a cytokine can be any cytokine.
  • a cytokine can be IL-2, IL-7, IL-12, IL-15, IL-21, or any combination thereof.
  • an artifical antigen presenting cell may be used to activate and/or expand a cell population.
  • an artifical may not induce allospecificity.
  • An aAPC may not express HLA in some cases.
  • An aAPC may be genetically modified to stably express genes that can be used to activation and/or stimulation.
  • a K562 cell may be used for activation.
  • a K562 cell may also be used for expansion.
  • a K562 cell can be a human erythroleukemic cell line.
  • a K562 cell may be engineered to express genes of interest.
  • K562 cells may not endogenously express HLA class I, II, or CDld molecules but may express ICAM-1 (CD54) and LFA-3 (CD58). K562 may be engineered to deliver a signal 1 to T cells. For example, K562 cells may be engineered to express HLA class I. In some cases, K562 cells may be engineered to express additional molecules such as B7, CD80, CD83, CD86, CD32, CD64, 4-1BBL, anti-CD3, anti-CD3 mAb, anti-CD28, anti-CD28mAb, CDld, anti- CD2, membrane -bound IL-15, membrane-bound IL-17, membrane-bound IL-21, membrane-bound IL-2, truncated CD 19, or any combination.
  • additional molecules such as B7, CD80, CD83, CD86, CD32, CD64, 4-1BBL, anti-CD3, anti-CD3 mAb, anti-CD28, anti-CD28mAb, CDld, anti- CD2, membrane -bound
  • an engineered K562 cell can expresses a membranous form of anti-CD3 mAb, clone OKT3, in addition to CD80 and CD83. In some cases, an engineered K562 cell can expresses a membranous form of anti-CD3 mAb, clone OKT3, membranous form of anti-CD28 mAb in addition to CD80 and CD83.
  • An aAPC can be a bead.
  • a spherical polystyrene bead can be coated with antibodies against CD3 and CD28 and be used for T cell activation.
  • a bead can be of any size. In some cases, a bead can be or can be about 3 and 6 micrometers. A bead can be or can be about 4.5 micrometers in size.
  • a bead can be utilized at any cell to bead ratio. For example, a 3 to 1 bead to cell ratio at 1 million cells per milliliter can be used.
  • An aAPC can also be a rigid spherical particle, a polystyrene latex microbeads, a magnetic nano- or micro-particles, a nanosized quantum dot, a 4, poly(lactic-co-glycolic acid) (PLGA) microsphere, a nonspherical particle, a 5, carbon nanotube bundle, a 6, ellipsoid PLGA microparticle, a 7, nanoworms, a fluidic lipid bilayer-containing system, an 8, 2D-supported lipid bilayer (2D-SLBs), a 9, liposome, a 10, RAFTsomes/microdomain liposome, an 11, SLB particle, or any combination thereof.
  • PLGA poly(lactic-co-glycolic acid)
  • an aAPC can expand CD4 T cells.
  • an aAPC can be engineered to mimic an antigen processing and presentation pathway of HLA class Il-restricted CD4 T cells.
  • a K562 can be engineered to express HLA-D, DP a, DP ⁇ chains, Ii, DM a, DM ⁇ , CD80, CD83, or any combination thereof.
  • engineered K562 cells can be pulsed with an HLA -restricted peptide in order to expand HLA -restricted antigen-specific CD4 T cells.
  • aAPCs can be combined with exogenously introduced cytokines for T cell activation, expansion, or any combination.
  • Cells can also be expanded in vivo, for example in the subject's blood after administration of genomically transplanted cells into a subject.
  • compositions and methods for intracellular genomic transplant can provide a cancer therapy with many advantages. For example, they can provide high efficiency gene transfer, expression, increased cell survival rates, an efficient introduction of recombinogenic double strand breaks, and a process that favors the Homology Directed Repair (HDR) over Non-Homologous End Joining (NHEJ) mechanism, and efficient recovery and expansion of homologous recombinants.
  • HDR Homology Directed Repair
  • NHEJ Non-Homologous End Joining
  • RNA ribonucleic acid
  • RNA ribonucleic acid
  • Cells to be engineered can be genetically modified with RNA or modified RNA instead of DNA to prevent DNA ⁇ e.g. , double or single stranded DNA) -induced toxicity and immunogenicity sometimes observed with the use of DNA.
  • a RNA/DNA fusion polynucleic acid can also be employed for genomic engineering.
  • RNA polynucleic acid system for gene editing of primary human T cells can be used, see e.g. FIG 5.
  • the schematic shows that an in vitro transcribed ribonucleic acid can be delivered and reverse transcribed into dsDNA inside a target cell.
  • a DNA template can then be used for a homologous recombination (HR) reaction inside the cell.
  • HR homologous recombination
  • robust genome engineering can be achieved by increasing the amount of polynucleic acid encoding a transgene. Introducing increased amounts of DNA may result in cellular toxicity, FIG. 2 and 3, in some cases; therefore it may be desirable to introduce RNA to a cell for genome engineering.
  • a transgene comprising an exogenous receptor sequence can be introduced into a cell for genome engineering via RNA, e.g., messenger RNA (mRNA).
  • RNA e.g., messenger RNA (mRNA).
  • mRNA messenger RNA
  • mRNA messenger RNA
  • One exemplary method utilizes in vitro transcription of a polynucleic acid to produce an mRNA polynucleic acid.
  • An mRNA polynucleic acid may then be transfected into a cell with a reverse transcriptase (RT) (either in protein form or a polynucleic acid encoding for a
  • RT reverse transcriptase
  • RT reverse transcriptase
  • RT reverse transcriptase
  • An RT protein is introduced into a cell.
  • An RT can be or can be derived from Avian Myeloblastosis Virus Reverse
  • AMV RT Moloney murine leukemia virus
  • M-MLV RT Moloney murine leukemia virus
  • HrV reverse transcriptase RT
  • derivatives thereof or combinations thereof may be transfected, a reverse transcriptase may transcribe the engineered mRNA polynucleic acid into a double strand
  • a reverse transcriptase can be an enzyme used to generate complementary DNA (cDNA) from an RNA template.
  • an RT enzyme can synthesize a complementary DNA strand initiating from a primer using RNA (cDNA synthesis) or single-stranded DNA as a template.
  • an RT may be functional at temperatures of 37 degrees Celsius. In other cases, an RT may be functional below temperatures of 37 degrees Celsius. In other cases, an RT may be functional at temperatures over 37 degrees Celsius.
  • An RT can be any enzyme that is used to generate complementary DNA (cDNA) from an RNA
  • An RT can be derived from retroviruses, hepatitis B virus, hepadnaviridae, or any double strand or single strand viruses.
  • An RT can have any number of biochemical activities.
  • an RT can have RNA-dependent DNA polymerase activity.
  • An RT can have ribonuclease H activity.
  • An RT can have DNA -dependent DNA polymerase activity.
  • an RT can be used to convert single-strand RNA to double strand cDNA. cDNA can subsequently be introduced into a cell genome.
  • FIG. 101A and FIG 101B shows in vivo reverse transcription of electroporated mRNA.
  • An RT can be an HIV-1 RT from human immunodeficiency virus type 1.
  • An HIV-1 RT can have subunits.
  • An HIV-1 RT can have two subunits.
  • An RT can also be from a Maloney murine leukemia virus (M-MLV).
  • M-MLV Maloney murine leukemia virus
  • An M-MLV virus may or may not have subunits.
  • a M-MLV virus is a single monomer.
  • An RT can also be an avian myeloblastosis virus RT (AMV RT).
  • An AMV RT may have subunits.
  • an AMV RT has two subunits.
  • a telomerase RT is also used.
  • a ds DNA can be used in a subsequent homologous recombination step.
  • a subsequent homologous recombination step can introduce an exogenous receptor sequence into the genome of a cell.
  • an introduced RT may need to be targeted to an introduced polynucleic acid.
  • introduced polynucleic acid may be RNA or DNA.
  • an introduced polynucleic acid may be a combination of RNA and DNA.
  • Targeting an introduced RT may be performed by incorporating a unique sequence to a polynucleic acid encoding for an engineered receptor, FIG 22. These unique sequences can help target the RT to a particular polynucleic acid.
  • a unique sequence can increase efficiency of a reaction.
  • Table 1 describes possible unique sequences to target an RT to an engineered polynucleic acid.
  • a unique sequence may be a sequence that may not be found in any human mRNA transcripts.
  • a unique sequence may be modified from a known mRNA transcript so that it is no longer an endogenous sequence.
  • a unique sequence may be identified using bioinformatics.
  • a unique sequence may be identified using publically available databases.
  • a unique sequence may be any base pair length in size.
  • a unique sequence can be or can be between 1-20 base pairs, 1-30 base pairs, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100 base pairs, or any length over 100 base pairs.
  • a unique sequence is or is between 1-20 base pairs.
  • a unique sequence is over 20 base pairs.
  • a unique sequence is exactly 20 base pairs in length.
  • a unique sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more base pairs.
  • multiple unique sequences are introduced into a polynucleic acid.
  • a unique sequence may be included in an engineered polynucleic acid.
  • sequence can be used to target a RT to an engineered polynucleic acid. In some cases,
  • oligonucleotides can be pre-annealed to an engineered polynucleic acid. Pre-annealed
  • oligonucleotides can encompass any length of a complementary unique sequence in an engineered polynucleic acid. Pre-annealed oligonucleotides may be exactly the same length of a complementary unique sequence or less than the entire length of a complementary sequence of a unique sequence in an engineered polynucleic acid,
  • a reverse transcriptase can be targeted to an engineered polynucleic acid by
  • a secondary structure can be any structure. In some cases, multiple secondary structures can be utilized.
  • a secondary structure can be a double helix.
  • a double helix can be formed by regions of many consecutive base pairs.
  • a double helix is a tertiary structure.
  • a double helix can be a spiral polymer.
  • a double helix can be right-handed.
  • a double helix can also be left-handed.
  • a double helix can be a right-handed structure that can contain a two nucleotide strand that can base pair together.
  • a single turn of a double helix can be or can be about 10 nucleotides.
  • a single turn of a double helix can be or can be about over 10 nucleotides or less than 10 nucleotides.
  • a single turn of a double helix can be or can be about 1-5 nucleotides, 1-10, 1-20, or over 20 nucleotides in length.
  • a secondary structure can also be a stem -loop or hairpin structure.
  • RNA hairpins can be formed when two complementary sequences in a single RNA molecule meet and bind together, after a folding or wrinkling of a molecule.
  • an RNA hairpin can consist of a double-stranded RNA (dsRNA) stem, and a terminal loop.
  • dsRNA double-stranded RNA
  • an RNA hairpin can occur in different positions within different types of RNAs.
  • An RNA hairpin may occur on a 5' end, a 3' end or anywhere in between a 5 ' end and a 3 ' end of a ribonucleic acid.
  • An RNA hairpin can differ in the length of a stem, the size of a loop, the number and size of bulges, and in the nucleotide sequence.
  • An RNA hairpin can be of any stem length.
  • An RNA hairpin can have any size loop.
  • a hairpin loop can be between or between about 4 to 8 bases long. In some cases, a hairpin loop is over or over about 8 bases long. In certain instances, a hairpin loop that is over or over about 8 bases long can have a secondary structure.
  • An RNA hairpin can have any number and size of bulges.
  • An RNA hairpin can be of any base pair length, e.g. , an RNA hairpin can be or can be about 1-100 base pairs, 1-200 base pairs, 1-300 base pairs, or over 300 base pairs.
  • An RNA hairpin can have secondary structure such as bulging for example.
  • an RNA hairpin can regulate gene expression in cis or trans, e.g. , an RNA hairpin
  • RNA molecules can regulate just that molecule (cis) or it can induce effects on other RNAs or pathways (trans).
  • Hairpins can serve as binding sites for a variety of proteins, act as substrates for enzymatic reactions as well as display intrinsic enzymatic activities.
  • a hairpin can be used to target an RT to an engineered polynucleic acid for transcription.
  • a hairpin structure can be located at a ribosome binding site.
  • a hairpin structure can facilitate translation.
  • a hairpin structure can have an internal ribosomal entry site (IRES).
  • IRES internal ribosomal entry site
  • An IRES sequence may allow for targeted transcription of an mRNA containing a hairpin.
  • a hairpin structure can direct an engineered polynucleic acid to a cellular location.
  • a hairpin can be or can contain a nuclear localization signal.
  • an RT can target an RNA hairpin of an engineered polynucleic acid.
  • An engineered polynucleic acid can contain one or more hairpin regions.
  • a hairpin can form at any location of an engineered polynucleic acid.
  • a secondary structure can also be a pseudoknot.
  • a pseudoknot can be a nucleic acid secondary
  • an H type pseudoknot can be used.
  • bases in a loop of a hairpin can form intramolecular pairs with bases outside of a stem. This can cause formation of a second stem and loop, resulting in a pseudoknot with two stems and two loops.
  • Two stems can be able to stack on top of each other to form a quasi-continuous helix with one continuous and one discontinuous strand.
  • a pseudoknot can be used to initiate transcription of an engineered polynucleic acid.
  • a pseudoknot can induce a ribosome to slip into alternative reading frames.
  • a pseudoknot can in some instances cause frame shifting.
  • an engineered polynucleic acid may need to be localized to a cellular nucleus.
  • An engineered polynucleic acid may encode for an exogenous or engineered receptor sequence that may need to be introduced into a genome of a cell.
  • introducing a receptor sequence to a cell genome may be performed by localizing an engineered polynucleic acid to a cell nuclease for transcription.
  • An engineered RNA polynucleic acid may be localized to a cellular nuclease. Localization may comprise any number of techniques.
  • a nuclear localization signal can be used to localize an engineered polynucleic acid encoding for an engineered receptor to a nucleus.
  • a nuclear localization signal can be any endogenous or engineered sequence.
  • a nuclear localization signal can be derived from a protein that may be strictly nuclear.
  • a protein that is nuclear may have nuclear localization signal that may not be affected by cellular state or its genomic expression locus.
  • nuclear localization may be derived from sequences or structures within a mature, spliced protein transcript.
  • a nuclear localization signal can be a BMP2-OP1 -responsive gene ("BORG")
  • BORG NLS sequences are included in a polynucleic acid. 1-5 BORG sequences may be included in some cases. In other cases, 5-10 BORG sequences are included. 1, 2,3,4,5,6,7,8,9, 10, or more BORG sequences can be included as a nuclear localization signal in a polynucleic acid. In some cases, as many BORG sequences that may be encoded within a polynucleic acid are utilized.
  • a nuclear localization signal can be a short, RNA motif consisting of a pentamer AGCCC with two sequence restrictions at positions -8 and -3 relative to the start of a pentamer.
  • a BORG sequence can be used in an engineered polynucleic acid to localize it to a nucleus of a cell.
  • a nuclear localization may be mediated by interaction of a SFl with tandem repeats of a short sequence that resembles the intronic branch site consensus sequence, resulting in localization of an RNA polynucleic acid to discrete nuclear subdomains.
  • a BORG sequence may function using by interacting with an abundant, nuclear-restricted protein or protein complex such as transcriptional complexes.
  • a nuclear localization sequence may interact with nuclear-localized RNAs or chromatin-associated RNA-protein complexes that may anchor a polynucleic acid containing a nuclear localization motif within the nucleus.
  • a nuclear localization sequence may interact with factors that can interfere with the formation of export complexes, resulting in retention of a polynucleic acid in a nucleus.
  • a nuclear localization signals may be a sequence, a structure, or any combination thereof.
  • nuclear localization of a nucleic acid may not require transport but only anchoring to a cytoskeleton (actin or intermediate filaments).
  • an engineered polynucleic acid can be transported on microtubules to a nucleus. Transport can take place in the form of large
  • ribonucleoprotein (RNP) complexes or RNP transport granules In some cases, a polynucleic acid can be complexed with a secondary protein that localizes it to a nucleus. In some cases, a transported polynucleic acid can be anchored at its final destination. Some trans-acting factors can shuttle back into the nucleus.
  • RNP ribonucleoprotein
  • a polynucleic acid may be introduced directly into a nucleus.
  • a polynucleic acid may be introduced directly into a nucleus.
  • polynucleic acid can be synthesized in a nucleus.
  • a polynucleic acid can be engineered to encode at least one BORG sequence.
  • a polynucleic acid can be engineered to encode for multiple BORG sequences.
  • a polynucleic acid can be engineered to encode for four BORG sequences.
  • a cell is transfected with a polynucleic acid containing a BORG sequence.
  • a polynucleic acid containing a BORG sequence can be localized into a cellular nucleus where it can participate in homologous recombination.
  • a polynucleic acid that is localized to a nucleus can encode a receptor sequence.
  • a receptor sequence can be introduced into a genome of a cell through a BORG-mediated nuclear localization.
  • polynucleic acids as described herein can be modified. A modification can be made at any one of
  • a polynucleic acid can undergo quality control after a modification.
  • quality control may include PAGE, HPLC, MS, or any combination thereof.
  • a modification can be a substitution, insertion, deletion, chemical modification, physical
  • a polynucleic acid can also be modified by 5 'adenylate, 5' guanosine-triphosphate cap, 5'N 7 -
  • Methylguanosine-triphosphate cap 5 'triphosphate cap, 3 'phosphate, 3 'thiophosphate, 5 'phosphate, 5 'thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9,3'-3' modifications, 5'-5' modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QS
  • 2'deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-0-methyl ribonucleoside analog, sugar modified analogs, wobble/universal bases, fluorescent dye label, 2'fluoro RNA, 2'O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5 ' -triphosphate, 5 -methylcytidine-5 ' -triphosphate, or any combination thereof.
  • a representative 2'O-methyl RNA modified gRNA is shown in FIG. 31.
  • a modification can be modification is permanent. In other cases, a modification is transient. In some cases, multiple modifications are made to a polynucleic acid.
  • a polynucleic acid modification may alter physico-chemical properties of a nucleotide, such as their conformation, polarity, hydrophobicity, chemical reactivity, base-pairing interactions, or any combination thereof.
  • a modification can also be a phosphorothioate substitute.
  • a natural phosphodiester bond may be susceptible to rapid degradation by cellular nucleases and; a modification of internucleotide linkage using phosphorothioate (PS) bond substitutes can be more stable towards hydrolysis by cellular degradation.
  • PS phosphorothioate
  • a modification can increase stability in a polynucleic acid.
  • a modification can also enhance biological activity.
  • a phosphorothioate enhanced RNA polynucleic acid can inhibit RNase A, RNase Tl, calf serum nucleases, or any combinations thereof.
  • PS-RNA polynucleic acids can be used in applications where exposure to nucleases is of high probability in vivo or in vitro.
  • phosphorothioate (PS) bonds can be introduced between the last 3-5 nucleotides at the 5'- or 3 '-end of a polynucleic acid which can inhibit exonuclease degradation.
  • phosphorothioate bonds can be added throughout an entire polynucleic acid to reduce attack by endonucleases.
  • a modification can be screened. Screening can include, but is not limited to, testing for immunogenicity, testing for toxicity, testing for efficiency of transcription, testing for efficiency of translation, or any combination thereof.
  • a modification may not be immunogenic.
  • a modification may not be toxic.
  • candidate modifications are screened prior to being incorporated into a polynucleic acid.
  • polynucleic acids with different modifications are screened to determine the level of immunogenicity, toxicity, efficacy, or any combination of the added modifications.
  • a modification is screened for its ability to support reverse transcription of a polynucleic acid.
  • a modification is a pseudouridine-5 ' -triphosphate (see e.g., FIG. 59). In other cases a modification is a 5 -methylcytidine-5 ' -triphosphate (see e.g., FIG. 59).
  • a modification can also include a change in chirality.
  • Polynucleic acids can be assembled by a variety of methods, e.g. , by automated solid-phase synthesis.
  • a polynucleic acid can be constructed using standard solid-phase DNA/RNA synthesis.
  • a polynucleic acid can also be constructed using a synthetic procedure.
  • a polynucleic acid can also be synthesized either manually or in a fully automated fashion.
  • a synthetic procedure may comprise 5'-hydroxyl oligonucleotides can be initially transformed into corresponding 5'-H- phosphonate mono esters, subsequently oxidized in the presence of imidazole to activated 5'- phosphorimidazolidates, and finally reacted with pyrophosphate on a solid support.
  • This procedure may include a purification step after the synthesis such as PAGE, HPLC, MS, or any combination thereof.
  • a polynucleic acid can be modified to make it less immunogenic and more stable for transfection into a cell.
  • a modified polynucleic acid can encode for any number of genes.
  • a polynucleic acid can encode for a transgene.
  • a transgene can encode for an engineered receptor.
  • a receptor can be a T cell receptor (TCR), B cell receptor (BCR), chimeric antigen receptor (CAR), or any combination thereof, see e.g., FIG. 57.
  • a receptor can be a TCR.
  • a modified polynucleic acid can be used in subsequent steps.
  • a modified polynucleic acid may be used in a homologous recombination reaction.
  • a homologous recombination reaction may include introducing a transgene encoding for an exogenous receptor in a genome of a cell.
  • An introduction may include any mechanism necessary to introduce a transgene sequence into a genome of a cell.
  • CRISPR is used in steps to introduce a receptor sequence into a genome of a cell.
  • Intracellular genomic transplant can be method of genetically modifying cells and nucleic acids for therapeutic applications.
  • the compositions and methods described throughout can use a nucleic acid- mediated genetic engineering process for tumor-specific TCR expression in a way that leaves the physiologic and immunologic anti-tumor potency of the T cells unperturbed.
  • Effective adoptive cell transfer-based immunotherapies can be useful to treat cancer (e.g., metastatic cancer) patients.
  • autologous peripheral blood lymphocytes can be modified using non-viral methods to express T Cell Receptors (TCR) that recognize unique mutations, neo-antigens, on cancer cells and can be used in the disclosed compositions and methods of an intracellular genomic transplant.
  • TCR T Cell Receptors
  • a cancer-specific TCR transgene can be inserted into the genome of a cell (e.g., T cell) using random or specific insertions.
  • the methods disclosed herein comprise introducing into the cell one or more nucleic acids (e.g., a first nucleic acid or a second acid).
  • a nucleic acid may generally refer to a substance whose molecules consist of many nucleotides linked in a long chain.
  • Non-limiting examples of the nucleic acid include an artificial nucleic acid analog (e.g., a peptide nucleic acid, a morpholino oligomer, a locked nucleic acid, a glycol nucleic acid, or a threose nucleic acid), a circular nucleic acid, a DNA, a single stranded DNA, a double stranded DNA, a genomic DNA, a plasmid, a plasmid DNA, a viral DNA, a viral vector, a gamma-retroviral vector, a lentiviral vector, an adeno-associated viral vector, an RNA, short hairpin RNA, psiRNA and/or a hybrid or combination thereof.
  • an artificial nucleic acid analog e.g., a peptide nucleic acid, a morpholino oligomer, a locked nucleic acid, a glycol nucleic acid, or a threose
  • a method may comprise a nucleic acid, and the nucleic acid is synthetic.
  • a sample may comprise a nucleic acid, and the nucleic acid may be fragmented.
  • a nucleic acid is a minicircle.
  • a nucleic acid may comprise promoter regions, barcodes, restriction sites, cleavage sites, endonuclease recognition sites, primer binding sites, selectable markers, unique identification sequences, resistance genes, linker sequences, or any combination thereof. In some aspects, these sites may be useful for enzymatic digestion, amplification, sequencing, targeted binding, purification, providing resistance properties (e.g., antibiotic resistance), or any combination thereof.
  • the nucleic acid may comprise one or more restriction sites.
  • a restriction site may generally refer to a specific peptide or nucleotide sequences at which site-specific molecules (e.g., proteases, endonucleases, or enzymes) may cut the nucleic acid.
  • a nucleic acid may comprise one or more restriction sites, wherein cleaving the nucleic acid at the restriction site fragments the nucleic acid.
  • the nucleic acid may comprise at least one endonuclease recognition site.
  • the endonuclease recognition site may comprise a Type I endonuclease recognition site, a Type II endonuclease recognition site, a Type III endonuclease recognition site, a Type IV endonuclease recognition site, or a Type V endonuclease recognition site.
  • Non-limiting examples of endonuclease recognition sites include an Aatll recognition site, an Acc65I recognition site, an Accl recognition site, an Acll recognition site, an
  • EcoRV recognition site an Fsel recognition site, an Fspl recognition site, an Haell recognition site, an
  • Hindi recognition site a Hindlll recognition site, an Hpal recognition site, a Kasl recognition site, a
  • Kpnl recognition site an Mfel recognition site, an Mlul recognition site, an Mscl recognition site, an
  • MspAlI recognition site an Mfel recognition site, an Mlul recognition site, an Mscl recognition site, an MspAlI recognition site, an Nael recognition site, a Narl recognition site, an Ncol recognition site, an Ndel recognition site, an NgoMIV recognition site, an Nhel recognition site, a Notl recognition site, an Nrul recognition site, an Nsil recognition site, an Nspl recognition site, a Pad recognition site, a Pcil recognition site, a Pmel recognition site, a Pmll recognition site, a Psil recognition site, a
  • PspOMI recognition site a Pstl recognition site, a Pvul recognition site, a PvuII recognition site, a
  • the restriction site may comprise Notl endonuclease recognition site.
  • a nucleic acid may readily bind to another nucleic acid (e.g., the nucleic acid comprises a sticky end or nucleotide overhang).
  • the nucleic acid may comprise an overhang at a first end of the nucleic acid.
  • a sticky end or overhang may refer to a series of
  • the nucleic acid may comprise a single stranded overhang at one or more ends of the nucleic acid.
  • the overhang can occur on the 3 ' end of the nucleic acid.
  • the overhang can occur on the 5 ' end of the nucleic acid.
  • the overhang can comprise any number of nucleotides.
  • the overhang can comprise 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, or 5 or more nucleotides.
  • the nucleic acid may require modification prior to binding to another nucleic acid (e.g., the nucleic acid may need to be digested with an endonuclease).
  • modification of the nucleic acid may generate a nucleotide overhang, and the overhang can comprise any number of nucleotides.
  • the overhang can comprise 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, or 5 or more nucleotides.
  • the nucleic acid may comprise a restriction site, wherein digesting the nucleic acid at the restriction site with a restriction enzyme (e.g., Notl) produces a 4 nucleotide overhang.
  • a restriction enzyme e.g., Notl
  • the modifying comprises generating a blunt end at one or more ends of the nucleic acid.
  • a blunt end may refer to a double stranded nucleic acid wherein both strands terminate in a base pair.
  • the nucleic acid may comprise a restriction site, wherein digesting the nucleic acid at the restriction site with a restriction enzyme (e.g., Bsal) produces a blunt end.
  • a restriction enzyme e.g., Bsal
  • Promoters are sequences of nucleic acid that control the binding of RNA polymerase and transcription factors, and can have a major effect on the efficiency of gene transcription, where a gene may be expressed in the cell, and/or what cell types a gene may be expressed in.
  • Non limiting examples of promoters include a cytomegalocirus (CMV) promoter, an elongation factor 1 alpha (EFla) promoter, a simian vacuolating virus (SV40) promoter, a phosphoglycerate kinase (PGK1) promoter, a ubiquitin C (Ubc) promoter, a human beta actin promoter, a CAG promoter, a Tetracycline response element (TRE) promoter, a UAS promoter, an Actin 5c (Ac5) promoter, a polyhedron promoter, Ca2+/calmodulin-dependent protein kinase II (CaMKIIa) promoter, a GAL1 promoter, a GAL 10 promoter, a TEFl promoter, a glyceraldehyde 3-phosphage dehydrogenase (GDS) promoter, an ADH1 promoter, a CaMV35S promoter, a Ubi promote
  • the nucleic acid may be a viral vector
  • the viral vector may comprise sequence encoding long terminal repeats (LTRs); U3-R-U5 regions found on either side of a retroviral provirus.
  • LTRs long terminal repeats
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding U3, a unique region at the 3' end of viral genomic RNA, containing sequences necessary for activation of viral genomic RNA transcription.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding R, a repeat region found within both the 5 'and
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding U5, a unique region at the 5' end of the viral genomic RNA.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding 5 ' LTR, which may acts as an RNA pol II promoter.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding a hybrid 5' LTR with a constitutive promoter such as CMV or RSV.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding a TAR, a trans-activating response element which may be located in the R region of the LTR and acts as a binding site for Tat.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding 3 ' LTR, which may be used to terminate trascription started by 5 ' LTR by the addition of a poly A tract following the R sequence.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding central polypurine tract (cPPT), a recognition site for proviral DNA synthesis. The presence of cPPT can affect transduction efficiency and transgene expression.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding Psi, an RNA target site for packaging by nucleocapsid.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding rev response element (RRE), a sequence to which the Rev protein binds.
  • RRE rev response element
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding the woodchuck hepatitis virus post-transcriptional regulatory element, a sequence that stimulates the expression of transgenes via increased nuclear export.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding GAG, a precursor structural protein of the lentiviral particle containing matrix, capsid, and nucleocapsid components.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding Pol, a precursor protein containing reverse transcriptase and integrase components.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding Rev, which may bind to RRE within unspliced and partially spliced transcripts to facilitate nuclear export.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding trans-activator (Tat), which may bind to TAR to activate transcription from the LTR promoter.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding vesicular stomatitis virus G glycoprotein (VSVG), a broad tropism envelope protein that can be used to psuedotype lentiviral vectors.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding inverted terminal repeat (ITR), which forms a T-shaped hairpin that can serve as the origin of viral DNA replication. ITR symmetry can affect the efficient multiplication of the AAV genome.
  • ITR inverted terminal repeat
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding Rep (e.g., Rep78, Rep68, Rep52, and Rep40), which are packaging proteins that are required for genome replication and necessary for integration.
  • the nucleic acid may be a viral vector, and the viral vector may comprise sequence encoding structural capsid proteins (e.g., VP1, VP2, and VP3), may serve to release the AAV particles from late endosomes and/or ensure correct virion assembly.
  • Rep e.g., Rep78, Rep68, Rep52, and Rep40
  • structural capsid proteins e.g., VP1, VP2, and VP3
  • the nucleic acid may comprise a barcode or a barcode sequence.
  • a barcode or barcode sequence relates to a natural or synthetic nucleic acid sequence comprised by a polynucleotide allowing for unambiguous identification of the polynucleotide and other sequences comprised by the polynucleotide having said barcode sequence.
  • a nucleic acid comprising a barcode can allow for identification of the encoded transgene.
  • a barcode sequence can comprise a sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 45, or 50 or more consecutive nucleotides.
  • a nucleic acid can comprise two or more barcode sequences or compliments thereof.
  • a barcode sequence can comprise a randomly assembled sequence of nucleotides.
  • a barcode sequence can be a degenerate sequence.
  • a barcode sequence can be a known sequence.
  • a barcode sequence can be a predefined sequence.
  • the methods disclosed herein may comprise a nucleic acid (e.g., a first nucleic acid and/or a second nucleic acid).
  • the nucleic acid may encode a transgene.
  • a transgene may refer to a linear polymer comprising multiple nucleotide subunits.
  • a transgene may comprise any number of nucleotides. In some cases, a transgene may comprise less than about 100 nucleotides.
  • a transgene may comprise at least about 100 nucleotides. In some cases, a transgene may comprise at least about 200 nucleotides. In some cases, a transgene may comprise at least about 300 nucleotides. In some cases, a transgene may comprise at least about 400 nucleotides. In some cases, a transgene may comprise at least about 500 nucleotides. In some cases, a transgene may comprise at least about 1000 nucleotides. In some cases, a transgene may comprise at least about 5000 nucleotides. In some cases, a transgene may comprise at least about 10,000 nucleotides. In some cases, a transgene may comprise at least about 20,000 nucleotides.
  • a transgene may comprise at least about 30,000 nucleotides. In some cases, a transgene may comprise at least about 40,000 nucleotides. In some cases, a transgene may comprise at least about 50,000 nucleotides. In some cases, a transgene may comprise between about 500 and about 5000 nucleotides. In some cases, a transgene may comprise between about 5000 and about 10,000 nucleotides. In any of the cases disclosed herein, the transgene may comprise DNA, RNA, or a hybrid of DNA and RNA. In some cases, the transgene may be single stranded. In some cases, the transgene may be double stranded, a. Random insertion
  • transgenes of the methods described herein can be inserted randomly into the genome of a cell. These transgenes can be functional if inserted anywhere in a genome. For instance, a transgene can encode its own promoter or can be inserted into a position where it is under the control of an endogenous promoter. Alternatively, a transgene can be inserted into a gene, such as an intron of a gene, an exon of a gene, a promoter, or a non-coding region.
  • a nucleic acid, e.g. , RNA, encoding a transgene sequences can be randomly inserted into a chromosome of a cell.
  • a random integration can result from any method of introducing a nucleic acid, e.g. , RNA, into a cell.
  • the method can be, but is not limited to, electroporation, sonoporation, use of a gene gun, lipotransfection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viral vectors including adenoviral, AAV, and retroviral vectors, and/or group II ribozymes.
  • a RNA encoding a transgene can also be designed to include a reporter gene so that the presence of a transgene or its expression product can be detected via activation of the reporter gene.
  • Any reporter gene can be used, such as those disclosed above. By selecting in cell culture those cells in which a reporter gene has been activated, cells can be selected that contain a transgene.
  • a transgene to be inserted can be flanked by engineered sites analogous to a targeted double strand break site in the genome to excise the transgene from a polynucleic acid so it can be inserted at the double strand break region.
  • a transgene can be virally introduced in some cases.
  • an AAV virus can be utilized to infect a cell with a transgene.
  • a modified or engineered AAV virus can be used to introduce a transgene to a cell, FIG. 83 A. and FIG. 83 B.
  • a modified or wildtype AAV can comprise homology arms to at least one genomic location, FIG. 84 to FIG. 86 D.
  • RNA encoding a transgene can be introduced into a cell via electroporation.
  • RNA can also be introduced into a cell via lipofection, infection, or transformation. Electroporation and/or lipofection can be used to transfect primary cells. Electroporation and/or lipofection can be used to transfect primary hematopoietic cells.
  • RNA can be reverse transcribed within a cell into DNA.
  • a DNA substrate can then be used in a homologous recombination reaction.
  • a DNA can also be introduced into a cell genome without the use of homologous recombination.
  • a DNA can be flanked by engineered sites that are complementary to the targeted double strand break region in a genome.
  • a DNA can be excised from a polynucleic acid so it can be inserted at a double strand break region without homologous recombination.
  • Expression of a transgene can be verified by an expression assay, for example, qPCR or by measuring levels of RNA.
  • Expression level can be indicative also of copy number. For example, if expression levels are extremely high, this can indicate that more than one copy of a transgene was integrated in a genome. Alternatively, high expression can indicate that a transgene was integrated in a highly transcribed area, for example, near a highly expressed promoter. Expression can also be verified by measuring protein levels, such as through Western blotting. In some cases, a splice acceptor assay can be used with a reporter system to measure transgene integration, FIG. 94.
  • Inserting one or more transgenes in any of the methods disclosed herein can be site-specific.
  • one or more transgenes can be inserted adjacent to or near a promoter.
  • one or more transgenes can be inserted adjacent to, near, or within an exon of a gene (e.g., PD-1 gene).
  • Such insertions can be used to knock-in a transgene (e.g., cancer-specific TCR transgene) while simultaneously disrupting another gene (e.g., PD-1 gene).
  • one or more transgenes can be inserted adjacent to, near, or within an intron of a gene.
  • a transgene can be introduced by an AAV viral vector and integrate into a targeted genomic location, FIG. 87.
  • Modification of a targeted locus of a cell can be produced by introducing DNA into cells, where the DNA has homology to the target locus.
  • DNA can include a marker gene, allowing for selection of cells comprising the integrated construct.
  • Complementary DNA in a target vector can recombine with a chromosomal DNA at a target locus.
  • a marker gene can be flanked by complementary DNA sequences, a 3' recombination arm, and a 5' recombination arm.
  • Multiple loci within a cell can be targeted. For example, transgenes with recombination arms specific to 1 or more target loci can be introduced at once such that multiple genomic modifications occur in a single step.
  • a variety of enzymes can catalyze insertion of foreign DNA into a host genome.
  • site-specific recombinases can be clustered into two protein families with distinct biochemical properties, namely tyrosine recombinases (in which DNA is covalently attached to a tyrosine residue) and serine recombinases (where covalent attachment occurs at a serine residue).
  • recombinases can comprise Cre, fC31 integrase (a serine recombinase derived from Streptomyces phage fC31), or bacteriophage derived site-specific recombinases (including Flp, lambda integrase, bacteriophage HK022 recombinase, bacteriophage R4 integrase and phage TP901-1 integrase).
  • Cre fC31 integrase
  • bacteriophage derived site-specific recombinases including Flp, lambda integrase, bacteriophage HK022 recombinase, bacteriophage R4 integrase and phage TP901-1 integrase.
  • Expression control sequences can also be used in constructs.
  • an expression control sequence can comprise a constitutive promoter, which is expressed in a wide variety of cell types.
  • Tissue-specific promoters can also be used and can be used to direct expression to specific cell lineages.
  • Site specific gene editing can be achieved using non-viral gene editing such as CRISPR, TALEN (see U.S. Pat. Nos. 14/193,037), transposon-based, ZEN, meganuclease, or Mega-TAL, or Transposon- based system.
  • non-viral gene editing such as CRISPR, TALEN (see U.S. Pat. Nos. 14/193,037), transposon-based, ZEN, meganuclease, or Mega-TAL, or Transposon- based system.
  • PiggyBac see Moriarty, B.S., et al, "Modular assembly of transposon integratable multigene vectors using RecWay assembly," Nucleic Acids Research (8):e92 (2013) or sleeping beauty
  • Aronovich, E.L, et al "The Sleeping Beauty transposon system: a non-viral vector for gene therapy," Hum. Mol. Genet., 20(R1): R14-R20. (2011) transpos
  • Site specific gene editing can also be achieved without homologous recombination.
  • An exogenous polynucleic acid can be introduced into a cell genome without the use of homologous recombination.
  • a transgene can be flanked by engineered sites that are complementary to a targeted double strand break region in a genome.
  • a transgene can be excised from a polynucleic acid so it can be inserted at a double strand break region without homologous recombination,
  • Transgenes can be useful for expressing, e.g. , overexpressing, endogenous genes at higher levels than without a transgenes. Additionally, transgenes can be used to express exogenous genes at a level greater than background, i.e., a cell that has not been transfected with a transgenes. Transgenes can also encompass other types of genes, for example, a dominant negative gene.
  • Transgenes can be placed into an organism, cell, tissue, or organ, in a manner which produces a
  • a polynucleic acid can comprise a transgene.
  • a polynucleic acid can encode an exogenous receptor, FIG. 57 A, FIG. 57 B, and FIG. 57 C.
  • TCR T cell receptor
  • a polynucleic acid comprising at least one exogenous T cell receptor (TCR) sequence flanked by at least two recombination arms having a sequence complementary to polynucleotides within a genomic sequence that is adenosine A2a receptor, CD276, V-set domain containing T cell activation inhibitor 1, B and T lymphocyte associated, cytotoxic T-lymphocyte-associated protein 4, indoleamine 2,3- dioxygenase 1, killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1, lymphocyte-activation gene 3, programmed cell death 1, hepatitis A virus cellular receptor 2, V- domain immunoglobulin suppressor of T-cell activation, or natural killer cell receptor 2B4.
  • TCR T cell receptor
  • a T cell can comprise one or more transgenes.
  • One or more transgenes can express a TCR alpha, beta, gamma, and/or delta chain protein recognizing and binding to at least one epitope (e.g., cancer epitope) on an antigen or bind to a mutated epitope on an antigen.
  • a TCR can bind to a cancer neo- antigen.
  • a TCR can be a functional TCR as shown in FIG. 22 and FIG. 26.
  • a TCR can comprise only one of the alpha chain or beta chain sequences as defined herein (e.g., in combination with a further alpha chain or beta chain, respectively) or may comprise both chains.
  • a TCR can comprise only one of the gamma chain or delta chain sequences as defined herein (e.g., in combination with a further gamma chain or delta chain, respectively) or may comprise both chains.
  • a functional TCR maintains at least substantial biological activity in the fusion protein.
  • this can mean that both chains remain able to form a T cell receptor (either with a non-modified alpha and/or beta chain or with another fusion protein alpha and/or beta chain) which exerts its biological function, in particular binding to the specific peptide-MHC complex of a TCR, and/or functional signal transduction upon peptide activation.
  • T cell receptor either with a non-modified gamma and/or delta chain or with another fusion protein gamma and/or delta chain
  • a T cell can also comprise one or more TCRs.
  • a T cell can also comprise a single TCRs specific to more than one target.
  • a TCR can be identified using a variety of methods.
  • a TCR can be identified using whole-exomic sequencing.
  • a TCR can target an ErbB2 interacting protein (ERBB2IP) antigen containing an E805G mutation identified by whole-exomic sequencing.
  • ERBB2IP ErbB2 interacting protein
  • a TCR can be identified from autologous, allogenic, or xenogeneic repertoires. Autologous and allogeneic identification can entail a multistep process. In both autologous and allogeneic identification, dendritic cells (DCs) can be generated from CD14-selected monocytes and, after maturation, pulsed or transfected with a specific peptide.
  • DCs dendritic cells
  • Peptide-pulsed DCs can be used to stimulate autologous or allogeneic T cells.
  • Single-cell peptide-specific T cell clones can be isolated from these peptide-pulsed T cell lines by limiting dilution.
  • TCRs of interest can be identified and isolated, a and ⁇ chains of a TCR of interest can be cloned, codon optimized, and encoded into a vector or transgene. Portions of a TCR can be replaced.
  • constant regions of a human TCR can be replaced with the corresponding murine regions. Replacement of human constant regions with corresponding murine regions can be performed to increase TCR stability.
  • a TCR can also be identified with high or supraphysiologic avidity ex vivo.
  • an appropriate target sequence should be identified.
  • the sequence may be found by isolation of a rare tumor-reactive T cell or, where this is not possible, alternative technologies can be employed to generate highly active anti-tumor T-cell antigens.
  • One approach can entail immunizing transgenic mice that express the human leukocyte antigen (HLA) system with human tumor proteins to generate T cells expressing TCRs against human antigens (see e.g., Stanislawski et al., Circumventing tolerance to a human MDM2 -derived tumor antigen by TCR gene transfer, Nature Immunology 2, 962 - 970 (2001)).
  • HLA human leukocyte antigen
  • An alternative approach can be allogeneic TCR gene transfer, in which tumor-specific T cells are isolated from a patient experiencing tumor remission and reactive TCR sequences can be transferred to T cells from another patient who shares the disease but may be non-responsive (de Witte, M. A., et al., Targeting self-antigens through allogeneic TCR gene transfer, Blood 108, 870-877(2006)).
  • in vitro technologies can be employed to alter a sequence of a TCR, enhancing their tumor-killing activity by increasing the strength of the interaction (avidity) of a weakly reactive tumor-specific TCR with target antigen (Schmid, D. A., et al., Evidence for a TCR affinity threshold delimiting maximal CD8 T cell function. J. Immunol. 184, 4936-4946 (2010)).
  • a TCR can be identified using whole-exomic sequencing.
  • the present functional TCR fusion protein can be directed against an MHC -presented epitope.
  • the MHC can be a class I molecule, for example HLA-A.
  • the MHC can be a class II molecule.
  • the present functional TCR fusion protein can also have a peptide-based or peptide-guided function in order to target an antigen.
  • the present functional TCR can be linked, for example, the present functional TCR can be linked with a 2A sequence.
  • the present functional TCR can also be linked with furin-V5-SGSGF2A as shown in FIG. 26.
  • the present functional TCR can also contain mammalian components.
  • the present functional TCR can contain mouse constant regions.
  • the present functional TCR can also in some cases contain human constant regions.
  • the peptide-guided function can in principle be achieved by introducing peptide sequences into a TCR and by targeting tumors with these peptide sequences.
  • These peptides may be derived from phage display or synthetic peptide library (see e.g., Arap, W., et al., "Cancer Treatment by Targeted Drug Delivery to Tumor Vasculature in a Mouse Model," Science, 279, 377-380 (1998); Scott, CP., et al, "Structural requirements for the biosynthesis of backbone cyclic peptide libraries," 8: 801-815 (2001)).
  • peptides specific for breast, prostate and colon carcinomas as well as those specific for neo-vasculatures were already successfully isolated and may be used in the present invention (Samoylova, T.I., et al., "Peptide Phage Display: Opportunities for Development of Personalized Anti -Cancer Strategies," Anti-Cancer Agents in Medicinal Chemistry, 6(1): 9-17(9) (2006)).
  • the present functional TCR fusion protein can be directed against a mutated cancer epitope or mutated cancer antigen.
  • Transgenes that can be used and are specifically contemplated can include those genes that exhibit a certain identity and/or homology to genes disclosed herein, for example, a TCR gene. Therefore, it is contemplated that if a gene exhibits at least or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,
  • a gene that exhibits at least or at least about 50%, 55%, 60%, 65%, 70%, 75%,
  • transgene can be used as a transgene.
  • the transgene can be functional.
  • Transgene can be incorporated into a cell.
  • a transgene can be incorporated into an
  • a transgene When inserted into a cell, a transgene can be either a complementary DNA (cDNA) segment, which is a copy of messenger RNA (mRNA), or a gene itself residing in its original region of genomic DNA (with or without introns).
  • cDNA complementary DNA
  • mRNA messenger RNA
  • a transgene of protein X can refer to a transgene comprising a nucleotide sequence encoding protein X.
  • a transgene encoding protein X can be a transgene encoding 100% or about 100% of the amino acid sequence of protein X.
  • a transgene encoding protein X can be a transgene encoding at least or at least about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the amino acid sequence of protein X.
  • transgene can ultimately result in a functional protein, e.g. , a partially, fully, or overly functional protein. As discussed above, if a partial sequence is expressed, the ultimate result can be a nonfunctional protein or a dominant negative protein. A nonfunctional protein or dominant negative protein can also compete with a functional (endogenous or exogenous) protein.
  • a transgene can also encode RNA (e.g., mRNA, shRNA, siRNA, or microRNA). In some cases, where a transgene encodes for an mRNA, this can in turn be translated into a polypeptide (e.g., a protein). Therefore, it is contemplated that a transgene can encode for protein.
  • a transgene can, in some instances, encode a protein or a portion of a protein. Additionally, a protein can have one or more mutations (e.g., deletion, insertion, amino acid replacement, or rearrangement) compared to a wild-type polypeptide.
  • a protein can be a natural polypeptide or an artificial polypeptide (e.g., a recombinant polypeptide).
  • a transgene can encode a fusion protein formed by two or more polypeptides.
  • a T cell can comprise or can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more transgenes.
  • a T cell can comprise one or more transgene comprising a TCR gene.
  • a transgene (e.g., TCR gene) can be inserted in a safe harbor locus.
  • a safe harbor can comprise a genomic location where a transgene can integrate and function without perturbing endogenous activity.
  • one or more transgenes can be inserted into any one of HPRT, AAVS SITE
  • transgene e.g., E.G. AAVS l, AAVS2, ETC.
  • CCR5 hROSA26
  • transgene e.g., E.G. AAVS l, AAVS2, ETC.
  • TCR gene can also be inserted in an endogenous immune checkpoint gene.
  • An endogenous immune checkpoint gene can be stimulatory checkpoint gene or an inhibitory checkpoint gene.
  • TCR gene e.g., TCR gene
  • a stimulatory checkpoint gene such as CD27, CD40, CD 122,
  • Immune checkpoint gene locations are provided using the
  • TCR gene e.g., TCR gene
  • A2AR endogenous inhibitory checkpoint gene
  • transgene can be inserted into any one of CD27, CD40, CD 122, OX40, GITR, CD 137,
  • a transgene can be inserted in an endogenous TCR gene.
  • a transgene can be inserted within a coding genomic region.
  • a transgene can also be inserted within a noncoding genomic region.
  • a transgene can be inserted into a genome without homologous recombination.
  • Insertion of a transgene can comprise a step of an intracellular genomic transplant.
  • a transgene can be inserted at a PD-1 gene, FIG. 46 A and FIG. 46 B.
  • more than one guide can target an immune checkpoint, FIG. 47.
  • a transgene can be integrated at a CTLA-4 gene, FIG. 48 and FIG. 50.
  • a transgene can be integrated at a CTLA-4 gene and a PD-1 gene, FIG. 49.
  • a transgene can also be integrated into a safe harbor such as AAVS 1, FIG. 96 and FIG. 97.
  • a transgene can be inserted into an AAV integration site.
  • An AAV integration site can be a safe harbor in some cases.
  • Alternative AAV integration sites may exist, such as AAVS2 on chromosome 5 or AAVS3 on chromosome 3. Additional AAV integration sites such as AAVS 2, AAVS3, AAVS4, AAVS5, AAVS6, AAVS7, AAVS8, and the like are also considered to be possible integration sites for an exogenous receptor, such as a TCR.
  • AAVS can refer to AAVS1 as well as related adeno-associated virus (AAVS) integration sites.
  • a chimeric antigen receptor can be comprised of an extracellular antigen recognition domain, a transmembrane domain, and a signaling region that controls T cell activation.
  • the extracellular antigen recognition domain can be derived from a murine, a humanized or fully human monoclonal antibody.
  • the extracellular antigen recognition domain is comprised of the variable regions of the heavy and light chains of a monoclonal antibody that is cloned in the form of single-chain variable fragments (scFv) and joined through a hinge and a transmembrane domain to an intracellular signaling molecule of the T-cell receptor (TCR) complex and at least one co-stimulatory molecule. In some cases a co-stimulatory domain is not used.
  • a CAR of the present disclosure can be present in the plasma membrane of a eukaryotic cell, e.g., a mammalian cell, where suitable mammalian cells include, but are not limited to, a cytotoxic cell, a T lymphocyte, a stem cell, a progeny of a stem cell, a progenitor cell, a progeny of a progenitor cell, and an NK cell.
  • a CAR can be active in the presence of its binding target.
  • a target can be expressed on a membrane.
  • a target can also be soluble (e.g., not bound to a cell).
  • a target can be present on the surface of a cell such as a target cell.
  • a target can be presented on a solid surface such as a lipid bilayer; and the like.
  • a target can be soluble, such as a soluble antigen.
  • a target can be an antigen.
  • An antigen can be present on the surface of a cell such as a target cell.
  • An antigen can be presented on a solid surface such as a lipid bilayer; and the like.
  • a target can be an epitope of an antigen.
  • a target can be a cancer neo-antigen.
  • a CAR can be comprised of a scFv targeting a tumor-specific neo-antigen.
  • a method can identify a cancer-related target sequence from a sample obtained from a cancer patient using an in vitro assay (e.g. whole-exomic sequencing).
  • a method can further identify a TCR transgene from a first T cell that recognizes the target sequence.
  • a cancer-related target sequence and a TCR transgene can be obtained from samples of the same patient or different patients.
  • a cancer- related target sequence can be encoded on a CAR transgene to render a CAR specific to a target sequence.
  • a method can effectively deliver a nucleic acid comprising a CAR transgene across a membrane of a T cell.
  • the first and second T cells can be obtained from the same patient.
  • the first and second T cells can be obtained from different patients. In other instances, the first and second T cells can be obtained from different patients.
  • the method can safely and efficiently integrate a CAR transgene into the genome of a T cell using a non-viral integration or a viral integration system to generate an engineered T cell and thus, a CAR transgene can be reliably expressed in the engineered T cell
  • a T cell can comprise one or more disrupted genes and one or more transgenes.
  • one or more genes whose expression is disrupted can comprise any one of CD27, CD40, CD 122, OX40, GITR, CD 137, CD28, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM- 3, PHD 1, PHD2, PHD 3, VISTA, CISH, PPP1R12C, and/or any combination thereof.
  • one or more genes whose expression is disrupted can comprise PD-land one or more transgenes comprise TCR.
  • one or more genes whose expression is disrupted can also comprise CTLA-4, and one or more transgenes comprise TCR.
  • a T cell can comprise one or more suppressed genes and one or more transgenes.
  • one or more genes whose expression is suppressed can comprise any one of CD27, CD40, CD 122, OX40, GITR, CD 137, CD28, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM- 3, PHD 1, PHD2, PHD 3, VISTA, CISH, PPP1R12C, and/or any combination thereof.
  • one or more genes whose expression is suppressed can comprise PD-1 and one or more transgenes comprise TCR.
  • one or more genes whose expression is suppressed can also comprise CTLA-4, and one or more transgenes comprise TCR.
  • a T cell can also comprise or can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • a dominant negative transgenes can suppress expression and/or function of a wild type counterpart of the dominant negative transgene.
  • a T cell comprising a dominant negative transgene X can have similar phenotypes compared to a different T cell comprising an X gene whose expression is suppressed.
  • One or more dominant negative transgenes can be dominant negative CD27, dominant negative CD40, dominant negative CD 122, dominant negative OX40, dominant negative GITR, dominant negative CD 137, dominant negative CD28, dominant negative ICOS, dominant negative A2AR, dominant negative B7- H3, dominant negative B7-H4, dominant negative BTLA, dominant negative CTLA-4, dominant negative IDO, dominant negative KIR, dominant negative LAG3, dominant negative PD-1, dominant negative TIM-3, dominant negative VISTA, dominant negative PHD1, dominant negative PHD2, dominant negative PHD3, dominant negative CISH, dominant negative CCR5, dominant negative HPRT, dominant negative AAVS SITE (E.G. AAVS 1, AAVS2, ETC.), dominant negative negative
  • PPP1R12C or any combination thereof.
  • RNAs that suppress genetic expression can comprise, but are not limited to, shRNA, siRNA, RNAi, and microRNA.
  • shRNA can be delivered to a T cell to suppress genetic expression.
  • a T cell can comprise one or more transgene encoding shRNAs.
  • shRNA can be specific to a particular gene.
  • a shRNA can be specific to any gene described in the application, including but not limited to, CD27, CD40, CD122, OX40, GITR, CD137, CD28, ICOS, A2AR, B7- H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, HPRT, AAVS SITE (E.G. AAVS1, AAVS2, ETC.), PHD1, PHD2, PHD3, CCR5, CISH, PPP1R12C, and/or any combination thereof.
  • One or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • one or more transgenes can be from different species.
  • transgene can be from a human, having a human genetic sequence.
  • One or more transgenes can comprise human genes. In some cases, one or more transgenes are not adenoviral genes.
  • a transgene can be inserted into a genome of a T cell in a random or site-specific manner, as
  • a transgene can be inserted to a random locus in a genome of a T cell.
  • These transgenes can be functional, e.g. , fully functional if inserted anywhere in a genome.
  • a transgene can encode its own promoter or can be inserted into a position where it is under the control of an endogenous promoter.
  • a transgene can be inserted into a gene, such as an intron of a gene or an exon of a gene, a promoter, or a non-coding region.
  • a transgene can be inserted such that the insertion disrupts a gene, e.g., an endogenous checkpoint.
  • a transgene insertion can comprise an endogenous checkpoint region.
  • a transgene insertion can be guided by
  • more than one copy of a transgene can be inserted into more than a random locus in a genome. For example, multiple copies can be inserted into a random locus in a genome. This can lead to increased overall expression than if a transgene was randomly inserted once.
  • a copy of a transgene can be inserted into a gene, and another copy of a transgene can be inserted into a different gene.
  • a transgene can be targeted so that it could be inserted to a specific locus in a genome of a T cell.
  • Expression of a transgene can be controlled by one or more promoters.
  • a promoter can be a ubiquitous, constitutive (unregulated promoter that allows for continual transcription of an associated gene), tissue-specific promoter or an inducible promoter. Expression of a transgene that is inserted adjacent to or near a promoter can be regulated. For example, a transgene can be inserted near or next to a ubiquitous promoter.
  • Some ubiquitous promoters can be a CAGGS promoter, an hCMV promoter, a PGK promoter, an SV40 promoter, or a ROSA26 promoter.
  • a promoter can be endogenous or exogenous.
  • one or more transgenes can be inserted adjacent or near to an endogenous or exogenous ROSA26 promoter.
  • a promoter can be specific to a T cell.
  • one or more transgenes can be inserted adjacent or near to a porcine ROSA26 promoter.
  • Tissue specific promoter or cell-specific promoters can be used to control the location of expression.
  • tissue-specific promoters can be a FABP promoter, a Lck promoter, a CamKII promoter, a CD 19 promoter, a Keratin promoter, an Albumin promoter, an aP2 promoter, an insulin promoter, an MCK promoter, an MyHC promoter, a WAP promoter, or a Col2A promoter.
  • Tissue specific promoter or cell-specific promoters can be used to control the location of expression.
  • tissue-specific promoters can be a FABP promoter, an Lck promoter, a CamKII promoter, a CD 19 promoter, a Keratin promoter, an Albumin promoter, an aP2 promoter, an insulin promoter, an MCK promoter, a MyHC promoter, a WAP promoter, or a Col2A promoter.
  • Inducible promoters can be used as well. These inducible promoters can be turned on and off when desired, by adding or removing an inducing agent. It is contemplated that an inducible promoter can be, but is not limited to, a Lac, tac, trc, trp, araBAD, phoA, recA, proU, cst-1, tetA, cadA, nar, PL, cspA, T7, VHB, Mx, and/or Trex.
  • an inducible promoter can be, but is not limited to, a Lac, tac, trc, trp, araBAD, phoA, recA, proU, cst-1, tetA, cadA, nar, PL, cspA, T7, VHB, Mx, and/or Trex.
  • a cell can be engineered to knock out endogenous genes.
  • Endogenous genes that can be knocked out can comprise immune checkpoint genes.
  • An immune checkpoint gene can be stimulatory checkpoint gene or an inhibitory checkpoint gene.
  • Immune checkpoint gene locations can be provided using the Genome Reference Consortium Human Build 38 patch release 2 (GRCh38.p2) assembly.
  • a gene to be knocked out can be selected using a database.
  • a database can comprise epigenetically permissive target sites.
  • a database can be ENCODE (encyclopedia of DNA Elements)
  • a databased can identify regions with open chromatin that can be more permissive to genomic engineering.
  • a T cell can comprise one or more disrupted genes. For example, one or more genes whose
  • adenosine A2a receptor CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO l), killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte- activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244), cytokine inducible SH2 -containing protein (CISH), hypoxanthine
  • ADORA adenosine A2a receptor
  • CD276 V-set domain containing T cell activation inhibitor 1
  • BTLA B and T lymphocyte associated
  • CTLA4 cytotoxic T-lymph
  • phosphoribosyltransferase 1 HPRT
  • AAVS SITE E.G. AAVS1, AAVS2, ETC.
  • CCR5 chemokine (C-C motif) receptor 5 (gene/pseudogene)
  • CD160 molecule CD 160
  • T-cell immunoreceptor with Ig and ITIM domains TAGIT
  • CD96 molecule CD96
  • CTAM cytotoxic and regulatory T-cell molecule
  • LAIRl leukocyte associated immunoglobulin like receptor l
  • SIGLEC7 sialic acid binding Ig like lectin 7
  • SIGLEC9 SIGLEC9
  • SIGLEC9 tumor necrosis factor receptor superfamily member 10b
  • TNFRSF10A tumor necrosis factor receptor superfamily member 10a
  • caspase 8 CASP8
  • caspase 10 caspase 10
  • CERP3 caspase 6
  • caspase 6 CASP6
  • SMAD2 SKI proto- oncogene
  • SKI SKI-like proto-oncogene
  • SKIL SKI-like proto-oncogene
  • TGFB induced factor homeobox l(TGIFl) interleukin 10 receptor subunit alpha
  • ILIORA interleukin 10 receptor subunit alpha
  • ILIORB heme oxygenase 2
  • HMOX2 interleukin 6 receptor
  • IL6ST interleukin 6 signal transducer
  • CSK c-src tyrosine kinase
  • PAG1 glycosphingolipid microdomains 1
  • SIT1 signaling threshold regulating transmembrane adaptor 1
  • FOXP3 PR domain l(PRDMl)
  • basic leucine zipper transcription factor ATF-like (BATF)
  • a T cell can comprise one or more suppressed genes.
  • one or more genes whose expression is suppressed can comprise any one of adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO l), killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte- activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244), cytokine inducible SH2 -containing protein (CISH), hypoxanthine
  • ADORA adenosine A2a receptor
  • HPRT phosphoribosyltransferase 1
  • AAVS SITE adeno-associated virus integration site
  • CCR5 chemokine (C-C motif) receptor 5 (gene/pseudogene) (CCR5), CD160 molecule (CD 160), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), CD96 molecule (CD96), cytotoxic and regulatory T-cell molecule (CRTAM), leukocyte associated immunoglobulin like receptor l(LAIRl), sialic acid binding Ig like lectin 7 (SIGLEC7), sialic acid binding Ig like lectin 9 (SIGLEC9), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member 10a (TNFRSF10A), caspase 8 (CASP8), caspase 10 (CASP10), caspase 3 (CASP3), caspase 6 (CASP6), caspase 7 (CASP7), Fas associated via death domain (FADD), Fas cell surface death receptor (FAS), transforming growth factor beta
  • SMAD2 SKI proto- oncogene
  • SKI SKI-like proto-oncogene
  • SKIL SKI-like proto-oncogene
  • TGFB induced factor homeobox l(TGIFl) interleukin 10 receptor subunit alpha
  • ILIORA interleukin 10 receptor subunit alpha
  • ILIORB heme oxygenase 2
  • HMOX2 interleukin 6 receptor
  • IL6ST interleukin 6 signal transducer
  • CSK c-src tyrosine kinase
  • PAG1 glycosphingolipid microdomains 1
  • SIT1 signaling threshold regulating transmembrane adaptor 1
  • FOXP3 PR domain l(PRDMl)
  • basic leucine zipper transcription factor ATF-like (BATF)
  • An engineered cell can target an antigen.
  • An engineered cell can also target an epitope.
  • An antigen can be a tumor cell antigen.
  • An epitope can be a tumor cell epitope.
  • Such a tumor cell epitope may be derived from a wide variety of tumor antigens such as antigens from tumors resulting from mutations (neo antigens or neo epitopes), shared tumor specific antigens, differentiation antigens, and antigens overexpressed in tumors.
  • Those antigens for example, may be derived from alpha-actinin-4,
  • ARTC1 BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4,
  • CDKN2A COA-1, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD, FN1, GPNMB, LDLR-fucosyltransferase fusion protein, HLA-A2d, HLA-A1 Id, hsp70-2, KIAAO205, MART2, ME1, MUM-lf, MUM-2, MUM-3, neo-PAP, Myosin class I, NFYC,
  • OGT OGT
  • OS-9 p53
  • pml-RARalpha fusion protein PRDX5
  • PTPRK PTPRK
  • K-ras K-ras
  • N-ras N-ras
  • SIRT2 SIRT2
  • SNRPDl SYT-SSX1- or -SSX2 fusion protein
  • TGF-betaRII TGF-betaRII
  • triosephosphate isomerase BAGE-1
  • GAGE-1 2, 8, Gage 3, 4, 5, 6, 7, GnTVf, HERV-K-MEL, KK-LC-1, KM-HN-1, LAGE-1, MAGE-
  • TRP2-INT2g TRP2-INT2g, XAGE-lb, CEA, gpl00/Pmell7, Kallikrein 4, mammaglobin-A, Melan- A/MART- 1 ,
  • Tumor-associated antigens may be antigens not normally expressed by the host; they can be mutated, truncated, misfolded, or otherwise abnormal manifestations of molecules normally expressed by the host; they can be identical to molecules normally expressed but expressed at abnormally high levels; or they can be expressed in a context or environment that is abnormal.
  • Tumor-associated antigens may be, for example, proteins or protein fragments, complex
  • carbohydrates gangliosides, haptens, nucleic acids, other biological molecules or any combinations thereof.
  • a target is a neo antigen or neo epitope.
  • a neo antigen can be a E805G mutation in ERBB2IP.
  • Neo antigen and neo epitopes can be identified by whole-exome sequencing in some cases.
  • a neo antigen and neo epitope target can be expressed by a gastrointestinal cancer cell in some cases.
  • a neo antigen and neo epitope can be expressed on an epithial carcinoma.
  • An epitope can be a stromal epitope. Such an epitope can be on the stroma of the tumor
  • the antigen can be a stromal antigen. Such an antigen can be on the stroma of the tumor microenvironment.
  • Those antigens and those epitopes can be present on tumor endothelial cells, tumor vasculature, tumor fibroblasts, tumor pericytes, tumor stroma, and/or tumor mesenchymal cells, just to name a few.
  • Those antigens for example, can comprise CD34, MCSP, FAP, CD31, PCNA, CD 1 17, CD40, MMP4, and/or Tenascin.
  • transgene can be done with or without the disruption of a gene.
  • a transgene can be inserted adjacent to, near, or within a gene such as CD27, CD40, CD 122, OX40, GITR, CD 137, CD28, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, HPRT, AAVS SITE (E.G. AAVS l, AAVS2, ETC.), CCR5, PPP1R12C, or CISH to reduce or eliminate the activity or expression of the gene.
  • a gene such as CD27, CD40, CD 122, OX40, GITR, CD 137, CD28, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, HPRT, AAVS SITE (E.G. AA
  • a cancer-specific TCR transgene can be inserted adjacent to, near, or within a gene (e.g. , PD-1) to reduce or eliminate the activity or expression of the gene.
  • a gene e.g. , PD-1
  • the insertion of a transgene can be done at an endogenous TCR gene.
  • the disruption of genes can be of any particular gene. It is contemplated that genetic homologues
  • genes that are disrupted can exhibit a certain identity and/or homology to genes disclosed herein, e.g., CD27, CD40, CD 122, OX40, GITR, CD 137, CD28, ICOS, A2AR, B7-H3, B7-H4,
  • BTLA CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, HPRT, CCR5, AAVS SITE (E.G.
  • a gene that exhibits or exhibits about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity (at the nucleic acid or protein level) can be disrupted.
  • Some genetic homologues are known in the art, however, in some cases, homologues are unknown. However, homologous genes between mammals can be found by comparing nucleic acid (DNA or R A) sequences or protein sequences using publically available databases such as NCBI BLAST.
  • a gene that can be disrupted can be a member of a family of genes.
  • a gene that can be disrupted can improve therapeutic potential of cancer immunotherapy.
  • a gene can be CISH.
  • a CISH gene can be a member of a cytokine-induced STAT inhibitor (CIS), also known as suppressor of cytokine signaling (SOCS) or STAT-induced STAT inhibitor (SSI), protein family (see e.g. , Palmer et al, Cish actively silences TCR signaling in CD 8+ T cells to maintain tumor tolerance, The Journal of Experimental Medicine 202(12), 2095-2113 (2015)).
  • CIS cytokine-induced STAT inhibitor
  • SOCS suppressor of cytokine signaling
  • SSI STAT-induced STAT inhibitor
  • a gene can be part of a SOCS family of proteins that can form part of a classical negative feedback system that can regulate cytokine signal transduction.
  • a gene to be disrupted can be CISH.
  • CISH can be involved in negative regulation of cytokines that signal through the JAK-STAT5 pathway such as erythropoietin, prolactin or interleukin 3 (IL-3) receptor.
  • a gene can inhibit STAT5 trans-activation by suppressing its tyrosine phosphorylation.
  • CISH family members are known to be cytokine -inducible negative regulators of cytokine signaling. Expression of a gene can be induced by IL2, IL3, GM-CSF or EPO in hematopoietic cells.
  • Proteasome-mediated degradation of a gene protein can be involved in the inactivation of an erythropoietin receptor.
  • a gene to be targeted can be expressed in tumor-specific T cells.
  • a gene to be targeted can increase infiltration of an engineered cell into antigen-relevant tumors when disrupted.
  • a gene to be targeted can be CISH.
  • a gene that can be disrupted can be involved in attenuating TCR signaling, functional avidity, or immunity to cancer. In some cases, a gene to be disrupted is upregulated when a TCR is stimulated. A gene can be involved in inhibiting cellular expansion, functional avidity, or cytokine
  • a gene can be involved in negatively regulating cellular cytokine production.
  • a gene can be involved in inhibiting production of effector cytokines, IFN-gamma and/or TNF for example.
  • a gene can also be involved in inhibiting expression of supportive cytokines such as IL-2 after TCR stimulation.
  • Such a gene can be CISH.
  • Gene suppression can also be done in a number of ways.
  • gene expression can be
  • RNA interfering reagents e.g., siRNA, shRNA, or microRNA.
  • a nucleic acid which can express shRNA can be stably transfected into a cell to knockdown expression.
  • a nucleic acid which can express shRNA can be inserted into the genome of a T cell, thus knocking down a gene within the T cell.
  • Disruption methods can also comprise overexpressing a dominant negative protein. This method can result in overall decreased function of a functional wild-type gene. Additionally, expressing a dominant negative gene can result in a phenotype that is similar to that of a knockout and/or knockdown.
  • a stop codon can be inserted or created (e.g., by nucleotide replacement), in one or more genes, which can result in a nonfunctional transcript or protein (sometimes referred to as knockout). For example, if a stop codon is created within the middle of one or more genes, the resulting transcription and/or protein can be truncated, and can be nonfunctional. However, in some cases, truncation can lead to an active (a partially or overly active) protein. If a protein is overly active, this can result in a dominant negative protein.
  • This dominant negative protein can be expressed in a nucleic acid within the control of any promoter.
  • a promoter can be a ubiquitous promoter.
  • a promoter can also be an inducible promoter, tissue specific promoter, cell specific promoter, and/or developmental specific promoter.
  • the nucleic acid that codes for a dominant negative protein can then be inserted into a cell. Any method can be used. For example, stable transfection can be used. Additionally, a nucleic acid that codes for a dominant negative protein can be inserted into a genome of a T cell.
  • One or more genes in a T cell can be knocked out or disrupted using any method. For example,
  • knocking out one or more genes can comprise deleting one or more genes from a genome of a T cell. Knocking out can also comprise removing all or a part of a gene sequence from a T cell. It is also contemplated that knocking out can comprise replacing all or a part of a gene in a genome of a T cell with one or more nucleotides. Knocking out one or more genes can also comprise inserting a sequence in one or more genes thereby disrupting expression of the one or more genes. For example, inserting a sequence can generate a stop codon in the middle of one or more genes. Inserting a sequence can also shift the open reading frame of one or more genes.
  • Knockout can be done in any cell, organ, and/or tissue, e.g., in a T cell, hematopoietic stem cell, in the bone marrow, and/or the thymus.
  • knockout can be whole body knockout, e.g. , expression of one or more genes is suppressed in all cells of a human.
  • Knockout can also be specific to one or more cells, tissues, and/or organs of a human. This can be achieved by conditional knockout, where expression of one or more genes is selectively suppressed in one or more organs, tissues or types of cells.
  • Conditional knockout can be performed by a Cre-lox system, wherein ere is expressed under the control of a cell, tissue, and/or organ specific promoter.
  • one or more genes can be knocked out (or expression can be suppressed) in one or more tissues, or organs, where the one or more tissues or organs can include brain, lung, liver, heart, spleen, pancreas, small intestine, large intestine, skeletal muscle, smooth muscle, skin, bones, adipose tissues, hairs, thyroid, trachea, gall bladder, kidney, ureter, bladder, aorta, vein, esophagus, diaphragm, stomach, rectum, adrenal glands, bronchi, ears, eyes, retina, genitals, hypothalamus, larynx, nose, tongue, spinal cord, or ureters, uterus, ovary, testis, and/or any combination thereof.
  • One or more genes can also be knocked out (or expression can be suppressed) in one types of cells, where one or more types of cells include trichocytes, keratinocytes, gonadotropes, corticotropes, thyrotropes, somatotropes, lactotrophs, chromaffin cells, parafollicular cells, glomus cells melanocytes, nevus cells, merkel cells, odontoblasts, cementoblasts corneal keratocytes, retina muller cells, retinal pigment epithelium cells, neurons, glias (e.g., oligodendrocyte astrocytes), ependymocytes, pineal ocytes, pneumocytes (e.g. , type I pneumocytes, and type II pneumocytes), clara cells, goblet cells, G cells, D cells,
  • glias e.g., oligodendrocyte astrocytes
  • pneumocytes
  • Enterochromaffin-like cells gastric chief cells, parietal cells, foveolar cells, K cells, D cells, I cells, goblet cells, paneth cells, enterocytes, microfold cells, hepatocytes, hepatic stellate cells (e.g. , Kupffer cells from mesoderm), cholecystocytes, centroacinar cells, pancreatic stellate cells, pancreatic a cells, pancreatic ⁇ cells, pancreatic ⁇ cells, pancreatic F cells, pancreatic ⁇ cells, thyroid (e.g.
  • follicular cells parathyroid (e.g., parathyroid chief cells), oxyphil cells, urothelial cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, myosatellite cells, tendon cells, cardiac muscle cells, lipoblasts, adipocytes, interstitial cells of cajal, angioblasts, endothelial cells, mesangial cells (e.g., intraglomerular mesangial cells and extraglomerular mesangial cells), juxtaglomerular cells, macula densa cells, stromal cells, interstitial cells, telocytes simple epithelial cells, podocytes, kidney proximal tubule brush border cells, Sertoli cells, leydig cells, granulosa cells, peg cells, germ cells, spermatozoon ovums, lymphocytes, myeloid cells,
  • the methods of the present disclosure may comprise obtaining one or more cells from a subject.
  • a cell may generally refer to any biological structure comprising cytoplasm, proteins, nucleic acids, and/or organelles enclosed within a membrane.
  • a cell may be a mammalian cell.
  • a cell may refer to an immune cell.
  • Non-limiting examples of a cell can include a B cell, a basophil, a dendritic cell, an eosinophil, a gamma delta T cell, a granulocyte, a helper T cell, a Langerhans cell, a lymphoid cell, an innate lymphoid cell (ILC), a macrophage, a mast cell, a megakaryocyte, a memory T cell, a monocyte, a myeloid cell, a natural killer T cell, a neutrophil, a precursor cell, a plasma cell, a progenitor cell, a regulatory T-cell, a T cell, a thymocyte, any differentiated or de-differentiated cell thereof, or any mixture or combination of cells thereof.
  • a B cell a basophil
  • a dendritic cell an eosinophil
  • a gamma delta T cell a granulocyte
  • a helper T cell a Langerhans cell
  • the cell may be an ILC, and the ILC is a group 1 ILC, a group 2 ILC, or a group 3 ILC.
  • Group 1 ILCs may generally be described as cells controlled by the T-bet transcription factor, secreting type-1 cytokines such as IFN-gamma and TNF -alpha in response to intracellular pathogens.
  • Group 2 ILCs may generally be described as cells relying on the GATA-3 and ROR-alpha transcription factors, producing type-2 cytokines in response to extracellular parasite infections.
  • Group 3 ILCs may generally be described as cells controlled by the ROR-gamma t transcription factor, and produce IL-17 and/or IL-22.
  • the cell may be a cell that is positive or negative for a given factor.
  • a cell may be a CD3+ cell, CD3- cell, a CD5+ cell, CD5- cell, a CD7+ cell, CD7- cell, a CD14+ cell, CD 14- cell, CD8+ cell, a CD8- cell, a CD 103+ cell, CD 103- cell, CD l lb+ cell, CD1 lb- cell, a BDCA1+ cell, a BDCA1- cell, an L-selectin+ cell, an L-selectin- cell, a CD25+, a CD25- cell, a CD27+, a CD27- cell, a CD28+ cell, CD28- cell, a CD44+ cell, a CD44- cell, a CD44- cell, a CD56+ cell, a CD56- cell, a CD57+ cell, a CD57- cell, a CD62L+ cell,
  • a cell may be positive or negative for any factor known in the art.
  • a cell may be positive for two or more factors.
  • a cell may be CD4+ and CD8+.
  • a cell may be negative for two or more factors.
  • a cell may be CD25-, CD44-, and CD69-.
  • a cell may be positive for one or more factors, and negative for one or more factors.
  • a cell may be CD4+ and CD8-. The selected cells can then be infused into a subject.
  • the cells may be selected for having or not having one or more given factors (e.g., cells may be separated based on the presence or absence of one or more factors). Separation efficiency can affect the viability of cells, and the efficiency with which a transgene may be integrated into the genome of a cell and/or expressed. In some
  • the selected cells can also be expanded in vitro.
  • the selected cells can be expanded in vitro prior to infusion.
  • cells used in any of the methods disclosed herein may be a mixture (e.g., two or more different cells) of any of the cells disclosed herein.
  • a method of the present disclosure may comprise cells, and the cells are a mixture of CD4+ cells and CD8+ cells.
  • a method of the present disclosure may comprise cells, and the cells are a mixture of CD4+ cells and naive cells.
  • Naive cells retain several properties that may be particularly useful for the methods disclosed herein.
  • naive cells are readily capable of in vitro expansion and T-cell receptor transgene expression, they exhibit fewer markers of terminal differentiation (a quality which may be associated with greater efficacy after cell infusion), and retain longer telomeres, suggestive of greater proliferative potential (Hinrichs, C.S., et al., "Human effector CD8+ T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy," Blood, 117(3):808-14
  • the methods disclosed herein may comprise selection or negative selection of markers specific for naive cells.
  • the cell may be a naive cell.
  • a naive cell may generally refer to any cell that has not been exposed to an antigen. Any cell in the present disclosure may be a naive cell.
  • a cell may be a naive T cell.
  • a naive T cell may generally be described a cell that has differentiated in bone marrow, and successfully undergone the positive and negative processes of central selection in the thymus, and/or may be characterized by the expression or absence of specific markers (e.g., surface expression of L-selectin, the absence of the activation
  • cells may comprise cell lines (e.g., immortalized cell lines).
  • cell lines include human BC-1 cells, human BJAB cells, human IM-9 cells, human Jiyoye cells, human K-562 cells, human LCL cells, mouse MPC-11 cells, human Raji cells, human Ramos cells, mouse Ramos cells, human RPMI8226 cells, human RS4-11 cells, human SKW6.4 cells, human Dendritic cells, mouse P815 cells, mouse RBL-2H3 cells, human HL-60 cells, human
  • NAMALWA cells human Macrophage cells, mouse RAW 264.7 cells, human KG-1 cells, mouse Ml cells, human PBMC cells, mouse BW5147 (T200-A)5.2 cells, human CCRF-CEM cells, mouse EL4 cells, human Jurkat cells, human SCID.adh cells, human U-937 cells or any combination of cells thereof.
  • Stem cells can give rise to a variety of somatic cells and thus have in principle the potential to serve as an endless supply of therapeutic cells of virtually any type.
  • the re-programmability of stem cells also allows for additional engineering to enhance the therapeutic value of the reprogrammed cell.
  • one or more cells may be derived from a stem cell.
  • Non- limiting examples of stem cells include embryonic stem cells, adult stem cells, tissue-specific stem cells, neural stem cells, allogenic stem cells, totipotent stem cells, multipotent stem cells, pluripotent stem cells, induced pluripotent stem cells, hematopoietic stem cells, epidermal stem cells, umbilical cord stem cells, epithelial stem cells, or adipose -derived stem cells.
  • a cell may be hematopoietic stem cell-derived lymphoid progenitor cells.
  • a cell may be embryonic stem cell -derived T cell.
  • a cell may be an induced pluripotent stem cell (iPSC)-derived T cell.
  • iPSC induced pluripotent stem cell
  • Conditional knockouts can be inducible, for example, by using tetracycline inducible promoters, development specific promoters. This can allow for eliminating or suppressing expression of a gene/protein at any time or at a specific time. For example, with the case of a tetracycline inducible promoter, tetracycline can be given to a T cell any time after birth.
  • a cre/lox system can also be under the control of a developmental specific promoter. For example, some promoters are turned on after birth, or even after the onset of puberty. These promoters can be used to control ere expression, and therefore can be used in developmental specific knockouts.
  • Knocking out technology can also comprise gene editing.
  • gene editing can be performed using a nuclease, including CRISPR associated proteins (Cas proteins, e.g. , Cas9), Zinc finger nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), and
  • Nucleases can be naturally existing nucleases, genetically modified, and/or recombinant.
  • Gene editing can also be performed using a transposon-based system (e.g. PiggyBac, Sleeping beauty).
  • gene editing can be performed using a transposase.
  • Methods described herein can take advantage of a CRISPR system.
  • CRISPR systems There are at least five types of CRISPR systems which all incorporate RNAs and Cas proteins.
  • Types I, III, and IV assemble a multi-Cas protein complex that is capable of cleaving nucleic acids that are complementary to the crRNA.
  • Types I and III both require pre-crRNA processing prior to assembling the processed crRNA into the multi-Cas protein complex.
  • Types II and V CRISPR systems comprise a single Cas protein complexed with at least one guiding RNA.
  • Site-specific cleavage of a target DNA occurs at locations determined by both 1) base- pairing complementarity between the guide RNA and the target DNA (also called a protospacer) and 2) a short motif in the target DNA referred to as the protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • an engineered cell can be generated using a CRISPR system, e.g., a type II CRISPR system.
  • a Cas enzyme used in the methods disclosed herein can be Cas9, which catalyzes DNA cleavage.
  • Enzymatic action by Cas9 derived from Streptococcus pyogenes or any closely related Cas9 can generate double stranded breaks at target site sequences which hybridize to 20 nucleotides of a guide sequence and that have a protospacer-adjacent motif (PAM) following the 20 nucleotides of the target sequence,
  • PAM protospacer-adjacent motif
  • a vector can be operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein (CRISPR-associated protein).
  • CRISPR enzyme such as a Cas protein (CRISPR-associated protein).
  • Cas proteins can include Casl,
  • CaslB Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl or Csxl2), CaslO, Csyl
  • An unmodified CRISPR enzyme can have DNA cleavage activity, such as Cas9.
  • a CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and/or within a complement of a target sequence.
  • a CRISPR enzyme can direct cleavage of one or both strands within or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • a vector that encodes a CRISPR enzyme that is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used.
  • a Cas protein can be a high fidelity cas protein such as Cas9HiFi.
  • a vector that encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs), such as more than or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, NLSs can be used.
  • a CRISPR enzyme can comprise more than or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, NLSs at or near the ammo-terminus, more than or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, NLSs at or near the carboxyl-terminus, or any combination of these (e.g. , one or more NLS at the ammo- terminus and one or more NLS at the carboxyl terminus).
  • each can be selected independently of others, such that a single NLS can be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies.
  • Cas9 can refer to a polypeptide with at least or at least about 50%, 60%, 70%, 80%, 90%, 100%
  • Cas9 can refer to a polypeptide with at most or at most about 50%, 60%, 70%, 80%, 90%, 100% sequence identity and/or sequence similarity to a wild type exemplary Cas9 polypeptide (e.g. , from S. pyogenes).
  • Cas9 can refer to the wild type or a modified form of the Cas9 protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.
  • a polynucleotide encoding an endonuclease (e.g., a Cas protein such as Cas9) can be codon optimized for expression in particular cells, such as eukaryotic cells. This type of optimization can entail the mutation of foreign-derived (e.g., recombinant) DNA to mimic the codon preferences of the intended host organism or cell while encoding the same protein.
  • CRISPR enzymes used in the methods can comprise NLSs.
  • the NLS can be located anywhere within the polypeptide chain, e.g., near the N- or C-terminus.
  • the NLS can be within or within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 amino acids along a polypeptide chain from the N- or C- terminus.
  • the NLS can be within or within about 50 amino acids or more, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 amino acids from the N- or C-terminus.
  • An endonuclease can comprise an amino acid sequence having at least or at least about 50%, 60%,
  • S. pyogenes Cas9 S. pyogenes Cas9 (SpCas9), Table 11, is commonly used as a CRISPR endonuclease for genome engineering, it may not be the best endonuclease for every target excision site.
  • the PAM sequence for SpCas9 (5' NGG 3') is abundant throughout the human genome, but a NGG sequence may not be positioned correctly to target a desired gene for modification.
  • a different endonuclease may be used to target certain genomic targets.
  • synthetic SpCas9-derived variants with non-NGG PAM sequences may be used.
  • other Cas9 orthologues from various species have been identified and these "non-SpCas9s" bind a variety of PAM sequences that could also be useful for the present invention.
  • the relatively large size of SpCas9 (approximately 4kb coding sequence) means that plasmids carrying the SpCas9 cDNA may not be efficiently expressed in a cell.
  • the coding sequence for Staphylococcus aureus Cas9 is approximately 1 kilo base shorter than SpCas9, possibly allowing it to be efficiently expressed in a cell. Similar to SpCas9, the SaCas9 endonuclease is capable of modifying target genes in mammalian cells in vitro and in mice in vivo.
  • Cas9 may include RNA-guided endonucleases from the Cpf 1 family that display cleavage activity in mammalian cells. Unlike Cas9 nucleases, the result of Cpf 1 -mediated DNA cleavage is a double-strand break with a short 3' overhang. Cpfl 's staggered cleavage pattern may open up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Like the Cas9 variants and orthologues described above, Cpfl may also expand the number of sites that can be targeted by CRISPR to AT- rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9.
  • any functional concentration of Cas protein can be introduced to a cell.
  • 15 micrograms of Cas mRNA can be introduced to a cell.
  • a Cas mRNA can be introduced from 0.5 micrograms to 100 micrograms.
  • a Cas mRNA can be introduced from 0.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 micrograms,
  • gRNA guide RNA
  • a guide RNA can comprise a guide sequence, or spacer sequence, that specifies a target site and guides an RNA/Cas complex to a specified target DNA for cleavage.
  • FIG. 15 demonstrates that guide RNA can target a CRISPR complex to three genes and perform a targeted double strand break.
  • Site-specific cleavage of a target DNA occurs at locations determined by both 1) base-pairing complementarity between a guide RNA and a target DNA (also called a protospacer) and 2) a short motif in a target DNA referred to as a protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • a method disclosed herein also can comprise introducing into a cell or embryo at least one guide
  • RNA or nucleic acid e.g., DNA encoding at least one guide RNA.
  • a guide RNA can interact with a
  • a guide RNA can comprise two RNAs, e.g., CRISPR RNA (crRNA) and transactivating crRNA
  • a guide RNA can sometimes comprise a single-guide RNA (sgRNA) formed by fusion of a portion (e.g., a functional portion) of crRNA and tracrRNA.
  • sgRNA single-guide RNA
  • a guide RNA can also be a dual RNA comprising a crRNA and a tracrRNA.
  • a guide RNA can comprise a crRNA and lack a tracrRNA.
  • a crRNA can hybridize with a target DNA or protospacer sequence.
  • a guide RNA can be an expression product.
  • a DNA that encodes a guide RNA can be a vector comprising a sequence coding for the guide RNA.
  • a guide RNA can be transferred into a cell or organism by transfecting the cell or organism with an isolated guide RNA or plasmid DNA comprising a sequence coding for the guide RNA and a promoter.
  • a guide RNA can also be transferred into a cell or organism in other way, such as using virus-mediated gene delivery.
  • a guide RNA can be isolated.
  • a guide RNA can be transfected in the form of an
  • a guide RNA can be prepared by in vitro transcription using any in vitro transcription system.
  • a guide RNA can be transferred to a cell in the form of isolated RNA rather than in the form of plasmid comprising encoding sequence for a guide RNA.
  • a guide RNA can comprise a DNA-targeting segment and a protein binding segment.
  • a DNA- targeting segment (or DNA-targeting sequence, or spacer sequence) comprises a nucleotide sequence that can be complementary to a specific sequence within a target DNA (e.g., a protospacer).
  • a protein-binding segment (or protein-binding sequence) can interact with a site-directed modifying polypeptide, e.g. an RNA -guided endonuclease such as a Cas protein.
  • segment it is meant a segment/section/region of a molecule, e.g., a contiguous stretch of nucleotides in an RNA.
  • a segment can also mean a region/section of a complex such that a segment may comprise regions of more than one molecule.
  • a protein-binding segment of a DNA-targeting RNA is one RNA molecule and the protein-binding segment therefore comprises a region of that RNA molecule.
  • the protein-binding segment of a DNA-targeting RNA comprises two separate molecules that are hybridized along a region of complementarity.
  • a guide RNA can comprise two separate RNA molecules or a single RNA molecule.
  • An exemplary single molecule guide RNA comprises both a DNA-targeting segment and a protein-binding segment.
  • An exemplary two-molecule DNA-targeting RNA can comprise a crRNA -like ("CRISPR RNA" or
  • targeter-RNA or “crRNA” or “crRNA repeat” molecule and a corresponding tracrRNA -like (“transacting CRISPR RNA” or “activator-RNA” or “tracrRNA”) molecule.
  • a first RNA molecule can be a crRNA -like molecule (targeter-RNA), that can comprise a DNA-targeting segment (e.g., spacer) and a stretch of nucleotides that can form one half of a double-stranded RNA (dsRNA) duplex comprising the protein-binding segment of a guide RNA.
  • dsRNA double-stranded RNA
  • a second RNA molecule can be a corresponding tracrRNA -like molecule (activator-RNA) that can comprise a stretch of nucleotides that can form the other half of a dsRNA duplex of a protein-binding segment of a guide RNA.
  • a stretch of nucleotides of a crRNA -like molecule can be complementary to and can hybridize with a stretch of nucleotides of a tracrRNA -like molecule to form a dsRNA duplex of a protein-binding domain of a guide RNA.
  • each crRNA-like molecule can be said to have a corresponding tracrRNA-like molecule.
  • a crRNA-like molecule additionally can provide a single stranded DNA-targeting segment, or spacer sequence.
  • a crRNA-like and a tracrRNA-like molecule (as a corresponding pair) can hybridize to form a guide RNA.
  • a subject two-molecule guide RNA can comprise any corresponding crRNA and tracrRNA pair.
  • a DNA-targeting segment or spacer sequence of a guide RNA can be complementary to sequence at a target site in a chromosomal sequence, e.g., protospacer sequence) such that the DNA-targeting segment of the guide RNA can base pair with the target site or protospacer.
  • a DNA- targeting segment of a guide RNA can comprise from or from about 10 nucleotides to from or from about 25 nucleotides or more.
  • a region of base pairing between a first region of a guide RNA and a target site in a chromosomal sequence can be or can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides in length.
  • a first region of a guide RNA can be or can be about 19, 20, or 21 nucleotides in length.
  • a guide RNA can target a nucleic acid sequence of or of about 20 nucleotides.
  • a target nucleic acid can be less than or less than about 20 nucleotides.
  • a target nucleic acid can be at least or at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.
  • a target nucleic acid can be at most or at most about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.
  • a target nucleic acid sequence can be or can be about 20 bases immediately 5' of the first nucleotide of the PAM.
  • a guide RNA can target the nucleic acid sequence.
  • a guide nucleic acid for example, a guide RNA, can refer to a nucleic acid that can hybridize to another nucleic acid, for example, the target nucleic acid or protospacer in a genome of a cell.
  • a guide nucleic acid can be RNA.
  • a guide nucleic acid can be DNA.
  • the guide nucleic acid can be programmed or designed to bind to a sequence of nucleic acid site-specifically.
  • a guide nucleic acid can comprise a polynucleotide chain and can be called a single guide nucleic acid.
  • a guide nucleic acid can comprise two polynucleotide chains and can be called a double guide nucleic acid.
  • a guide nucleic acid can comprise one or more modifications to provide a nucleic acid with a new or enhanced feature.
  • a guide nucleic acid can comprise a nucleic acid affinity tag.
  • a guide nucleic acid can comprise synthetic nucleotide, synthetic nucleotide analog, nucleotide derivatives, and/or modified nucleotides.
  • a guide nucleic acid can comprise a nucleotide sequence (e.g. , a spacer), for example, at or near the 5' end or 3' end, that can hybridize to a sequence in a target nucleic acid (e.g. , a protospacer).
  • a spacer of a guide nucleic acid can interact with a target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing).
  • a spacer sequence can hybridize to a target nucleic acid that is located 5' or 3' of a protospacer adjacent motif (PAM).
  • the length of a spacer sequence can be at least or at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.
  • a guide RNA can also comprises a dsRNA duplex region that forms a secondary structure.
  • a secondary structure formed by a guide RNA can comprise a stem (or hairpin) and a loop.
  • a length of a loop and a stem can vary.
  • a loop can range from about 3 to about 10 nucleotides in length
  • a stem can range from about 6 to about 20 base pairs in length.
  • a stem can comprise one or more bulges of 1 to about 10 nucleotides.
  • the overall length of a second region can range from about 16 to about 60 nucleotides in length.
  • a loop can be or can be about 4 nucleotides in length and a stem can be or can be about 12 base pairs.
  • a dsRNA duplex region can comprise a protein-binding segment that can form a complex with an RNA -binding protein, such as a RNA-guided endonuclease, e.g. Cas protein.
  • a guide RNA can also comprise a tail region at the 5' or 3' end that can be essentially single-stranded.
  • a tail region is sometimes not complementarity to any chromosomal sequence in a cell of interest and is sometimes not complementarity to the rest of a guide RNA.
  • the length of a tail region can vary.
  • a tail region can be more than or more than about 4 nucleotides in length.
  • the length of a tail region can range from or from about 5 to from or from about 60 nucleotides in length.
  • a guide RNA can be introduced into a cell or embryo as an RNA molecule.
  • a RNA molecule can be transcribed in vitro and/or can be chemically synthesized.
  • a guide RNA can then be introduced into a cell or embryo as an RNA molecule.
  • a guide RNA can also be introduced into a cell or embryo in the form of a non-RNA nucleic acid molecule, e.g., DNA molecule.
  • a DNA encoding a guide RNA can be operably linked to promoter control sequence for expression of the guide RNA in a cell or embryo of interest.
  • a RNA coding sequence can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol III).
  • a DNA molecule encoding a guide RNA can also be linear.
  • a DNA molecule encoding a guide RNA can also be circular.
  • a DNA sequence encoding a guide RNA can also be part of a vector.
  • vectors can include plasmid vectors, phagemids, cosmids, artificial/mini-chromosomes, transposons, and viral vectors.
  • a DNA encoding a RNA-guided endonuclease is present in a plasmid vector.
  • suitable plasmid vectors include pUC, pBR322, pET, pBluescript, and variants thereof.
  • a vector can comprise additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc.), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like.
  • additional expression control sequences e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc.
  • selectable marker sequences e.g., antibiotic resistance genes
  • a Cas protein such as a Cas9 protein or any derivative thereof, can be pre-complexed with a guide RNA to form a ribonucleoprotein (RNP) complex.
  • the RNP complex can be introduced into primary immune cells. Introduction of the RNP complex can be timed. The cell can be synchronized with other cells at Gl, S, and/or M phases of the cell cycle. The RNP complex can be delivered at a cell phase such that HDR is enhanced. The RNP complex can facilitate homology directed repair.
  • a guide RNA can also be modified.
  • the modifications can comprise chemical alterations, synthetic modifications, nucleotide additions, and/or nucleotide subtractions.
  • the modifications can also enhance CRISPR genome engineering.
  • a modification can alter chirality of a gRNA. In some cases, chirality may be uniform or stereopure after a modification.
  • a guide RNA can be synthesized. The synthesized guide RNA can enhance CRISPR genome engineering.
  • a guide RNA can also be truncated. Truncation can be used to reduce undesired off-target mutagenesis. The truncation can comprise any number of nucleotide deletions.
  • the truncation can comprise 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more nucleotides.
  • a guide RNA can comprise a region of target complementarity of any length.
  • a region of target complementarity can be less than 20 nucleotides in length.
  • a region of target complementarity can be more than 20 nucleotides in length.
  • a dual nickase approach may be used to introduce a double stranded break.
  • nickase proteins can be mutated at known amino acids within either nuclease domains, thereby deleting activity of one nuclease domain and generating a nickase Cas protein capable of generating a single strand break.
  • a nickase along with two distinct guide RNAs targeting opposite strands may be utilized to generate a DSB within a target site (often referred to as a "double nick” or "dual nickase” CRISPR system). This approach may dramatically increase target specificity, since it is unlikely that two off- target nicks will be generated within close enough proximity to cause a DSB.
  • GUIDE-Seq analysis can be performed to determine the specificity of engineered guide RNAs.
  • the general mechanism and protocol of GUIDE-Seq profiling of off-target cleavage by CRISPR system nucleases is discussed in Tsai, S. et al, "GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR system nucleases," Nature, 33: 187-197 (2015).
  • a gRNA can be introduced at any functional concentration.
  • a gRNA can be introduced to a cell at lOmicrograms.
  • a gRNA can be introduced from 0.5 micrograms to 100 micrograms.
  • a gRNA can be introduced from 0.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 micrograms.
  • a method can comprise an endonuclease selected from the group consisting of Casl,
  • a Cas protein can be Cas9.
  • a method can further comprise at least one guide RNA (gRNA).
  • gRNA can comprise at least one modification.
  • An exogenous TCR can bind a cancer neo-antigen.
  • Disclsoed herein is a method of making an engineered cell comprising: introducing at least one polynucleic acid encoding at least one exogenous T cell receptor (TCR) receptor sequence;
  • gR A guide RNA
  • gR A guide RNA
  • endonuclease wherein the gRNA comprises at least one sequence complementary to at least one endogenous genome.
  • a modification is on a 5' end, a 3' end, from a 5' end to a 3' end, a single base modification, a 2'-ribose modification, or any combination thereof.
  • a modification can be selected from a group consisting of base substitutions, insertions, deletions, chemical modifications, physical modifications, stabilization, purification, and any combination thereof.
  • a modification is a chemical modification.
  • a modification can be selected from
  • DABCYL SE dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linkers, 2'deoxyribonucleoside analog purine, 2'deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-0-methyl ribonucleoside analog, sugar modified analogs, wobble/universal bases, fluorescent dye label, 2'fluoro RNA, 2'O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'- triphosphate, 5-methylcytidine-5'-triphosphate, 2-O-methyl 3phosphorothioate or any combinations thereof.
  • a modification can be a pseudouride modification as shown in FIG. 98. In some cases, a modification may not affect viability, FIG.
  • a modification is a 2-O-methyl 3 phosphorothioate addition.
  • a 2-O-methyl 3 phosphorothioate addition is a 2-O-methyl 3 phosphorothioate addition.
  • phosphorothioate addition can be performed from 1 base to 150 bases.
  • phosphorothioate addition can be performed from 1 base to 4 bases.
  • a 2-O-methyl 3 phosphorothioate addition can be performed on 2 bases.
  • a 2-O-methyl 3 phosphorothioate addition can be performed on 4 bases.
  • a modification can also be a truncation.
  • a truncation can be a 5 base truncation.
  • a 5 base truncation can prevent a Cas protein from performing a cut.
  • An endonuclease can be selected from the group consisting of a CRISPR system, TALEN, Zinc Finger, transposon- based, ZEN, meganuclease, Mega-TAL, and any combination.
  • An endonuclease can be a Cas endonuclease.
  • a Cas endonuclease can be selected from the group consisting of Casl, Cas IB, Cas2,
  • a Cas9HiFi homologues thereof or modified versions thereof.
  • a modififed version of a Cas can be a clean cas, as shown in FIG. 100 A and B.
  • a Cas protein can be Cas9.
  • a Cas9 can create a double strand break in said at least one endogenous genome.
  • an endogenous genome comprises at least one gene.
  • a gene can be CISH, PD-1, TRA, TRB, or a combination thereof.
  • a double strand break can be repaired using homology directed repair (HR), nonhomologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or any combination or derivative thereof.
  • a TCR can be integrated into a double strand break,
  • transgene e.g., exogenous sequence
  • a transgene is typically not identical to the genomic sequence where it is placed.
  • a donor transgene can contain a non-homologous sequence flanked by two regions of homology to allow for efficient HDR at the location of interest.
  • transgene sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin.
  • a transgene can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, a sequence can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest.
  • a transgene polynucleic acid can be DNA or RNA, single-stranded or double-stranded and can be introduced into a cell in linear or circular form.
  • a transgene sequence(s) can be contained within a DNA mini -circle, which may be introduced into the cell in circular or linear form. If introduced in linear form, the ends of a transgene sequence can be protected (e.g., from exonucleolytic degradation) by any method. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and/or self-complementary oligonucleotides can be ligated to one or both ends.
  • Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.
  • a transgene can be flanked by recombination arms.
  • recombination arms can be flanked by recombination arms.
  • a transgene can also be integrated into a genomic region such that the insertion disrupts an endogenous gene.
  • a transgene can be integrated by any method, e.g., non-recombination end joining and/or recombination directed repair.
  • a transgene can also be integrated during a recombination event where a double strand break is repaired.
  • a transgene can also be integrated with the use of a homologous recombination enhancer. For example, an enhancer can block non-homologous end joining so that homology directed repair is performed to repair a double strand break.
  • a transgene can be flanked by recombination arms where the degree of homology between the arm and its complementary sequence is sufficient to allow homologous recombination between the two.
  • the degree of homology between the arm and its complementary sequence can be 50% or greater.
  • Two homologous non-identical sequences can be any length and their degree of non- homology can be as small as a single nucleotide (e.g., for correction of a genomic point mutation by targeted homologous recombination) or as large as 10 or more kilobases (e.g., for insertion of a gene at a predetermined ectopic site in a chromosome).
  • Two polynucleotides comprising the homologous non-identical sequences need not be the same length.
  • a representative transgene with recombination arms to CCR5 is shown in FIG. 16. Any other gene, e.g., the genes described herein, can be used to generate a recombination arm.
  • a transgene can be flanked by engineered sites that are complementary to the targeted double strand break region in a genome. In some cases, engineered sites are not recombination arms. Engineered sites can have homology to a double strand break region. Engineered sites can have homology to a gene. Engineered sites can have homology to a coding genomic region. Engineered sites can have homology to a non-coding genomic region. In some cases, a transgene can be excised from a polynucleic acid so it can be inserted at a double strand break region without homologous recombination. A transgene can integrate into a double strand break without homologous recombination.
  • a polynucleotide can be introduced into a cell as part of a vector molecule having additional
  • transgene polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)).
  • viruses e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)
  • a virus that can deliver a transgene can be an AAV virus.
  • a transgene is generally inserted so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the endogenous gene into which a transgene is inserted (e.g., AAVS SITE (E.G. AAVS1, AAVS2, ETC.), CCR5, HPRT).
  • a transgene may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue/cell specific promoter.
  • a minicircle vector can encode a transgene.
  • Targeted insertion of non-coding nucleic acid sequence may also be achieved. Sequences encoding antisense RNAs, RNAi, shRNAs and micro RNAs (miRNAs) may also be used for targeted insertions.
  • a transgene may be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed.
  • a transgene as described herein can be inserted into an endogenous locus such that some (N-terminal and/or C-terminal to a transgene) or none of the endogenous sequences are expressed, for example as a fusion with a transgene.
  • a transgene e.g., with or without additional coding sequences such as for the endogenous gene
  • a TCR transgene can be inserted into an endogenous TCR gene. For example, FIG.
  • transgene can be inserted into an endogenous CCR5 gene.
  • a transgene can be inserted into any gene, e.g., the genes as described herein.
  • the endogenous sequences can be full-length sequences (wild-type or mutant) or partial sequences.
  • the endogenous sequences can be functional. Non-limiting examples of the function of these full length or partial sequences include increasing the serum half-life of the polypeptide expressed by a transgene (e.g., therapeutic gene) and/or acting as a carrier.
  • exogenous sequences may also include
  • transcriptional or translational regulatory sequences for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
  • the exogenous sequence (e.g., transgene) comprises a fusion of a protein of interest and, as its fusion partner, an extracellular domain of a membrane protein, causing the fusion protein to be located on the surface of the cell.
  • a transgene encodes a TCR wherein a TCR encoding sequence is inserted into a safe harbor such that a TCR is expressed.
  • a TCR encoding sequence is inserted into a PD 1 and/or a CTLA-4 locus.
  • a TCR is delivered to the cell in a lentivirus for random insertion while the PD1- or CTLA-4 specific nucleases can be supplied as mRNAs.
  • a TCR is delivered via a viral vector system such as a retrovirus, AAV or adenovirus along with mRNA encoding nucleases specific for a safe harbor (e.g. AAVS SITE (E.G. AAVS 1, AAVS2, ETC.), CCR5, albumin or HPRT).
  • a viral vector system such as a retrovirus, AAV or adenovirus along with mRNA encoding nucleases specific for a safe harbor (e.g. AAVS SITE (E.G. AAVS 1, AAVS2, ETC.), CCR5, albumin or HPRT).
  • the cells can also be treated with mRNAs encoding PD 1 and/or CTLA-4 specific nucleases.
  • the polynucleotide encoding a TCR is supplied via a viral delivery system together with mRNA encoding HPRT specific nucleases and PD 1- or CTLA-4 specific nucleases.
  • Cells comprising an integrated TCR-encoding nucleotide at the HPRT locus can be selected for using 6-thioguanine, a guanine analog that can result in cell arrest and/or initiate apoptosis in cells with an intact HPRT gene.
  • TCRs that can be used with the methods and compositions of the invention include all types of these chimeric proteins, including first, second and third generation designs.
  • TCRs comprising specificity domains derived from antibodies can be particularly useful, although specificity domains derived from receptors, ligands and engineered polypeptides can be also envisioned by the invention.
  • the intercellular signaling domains can be derived from TCR chains such as zeta and other members of the CD3 complex such as the ⁇ and E chains.
  • a TCRs may comprise additional co-stimulatory domains such as the intercellular domains from CD28, CD 137 (also known as 4-1BB) or CD 134.
  • additional co-stimulatory domains such as the intercellular domains from CD28, CD 137 (also known as 4-1BB) or CD 134.
  • two types of co-stimulator domains may be used simultaneously (e.g., CD3 zeta used with
  • CD28+CD 137 CD28+CD 137
  • the engineered cell can be a stem memory TSCM cell comprised of CD45RO (-),
  • stem memory cells can also express CD95, IL-2R , CXCR3, and LFA- 1, and show numerous functional attributes distinctive of stem memory cells.
  • Engineered cells can also be central memory TCM cells comprising L-selectin and CCR7, where the central memory cells can secrete, for example, IL-2, but not IFNy or IL-4.
  • Engineered cells can also be effector memory TEM cells comprising L-selectin or CCR7 and produce, for example, effector cytokines such as ⁇ and IL-4.
  • a population of cells can be introduced to a subject.
  • a population of cells can be a combination of T cells and NK cells.
  • a population can be a combination of naive cells and effector cells.
  • a homologous recombination HR enhancer can be used to suppress non-homologous end-joining (NHEJ).
  • NHEJ non-homologous end-joining
  • Non-homologous end-joining can result in the loss of nucleotides at the end of double stranded breaks; non-homologous end-joining can also result in frameshift. Therefore, homology-directed repair can be a more attractive mechanism to use when knocking in genes.
  • a HR enhancer can be delivered. In some cases, more than one HR enhancer can be delivered.
  • a HR enhancer can inhibit proteins involved in non-homologous end-joining, for example, KU70, KU80, and/or DNA Ligase IV.
  • a Ligase IV inhibitor such as Scr7
  • the HR enhancer can be L755507.
  • a different Ligase IV inhibitor can be used.
  • a HR enhancer can be an adenovirus 4 protein, for example, E1B55K and/or E4orf6.
  • a chemical inhibitor can be used.
  • Non-homologous end-joining molecules such as KU70, KU80, and/or DNA Ligase IV can be
  • non-homologous end-joining molecules such as KU70, KU80, and/or DNA Ligase IV can be suppressed by gene silencing.
  • nonhomologous end-joining molecules KU70, KU80, and/or DNA Ligase IV can be suppressed by gene silencing during transcription or translation of factors.
  • Non-homologous end-joining molecules KU70, KU80, and/or DNA Ligase IV can also be suppressed by degradation of factors.
  • Nonhomologous end-joining molecules KU70, KU80, and/or DNA Ligase IV can be also be inhibited.
  • Inhibitors of KU70, KU80, and/or DNA Ligase IV can comprise E1B55K and/or E4orf6.
  • Nonhomologous end-joining molecules KU70, KU80, and/or DNA Ligase IV can also be inhibited by sequestration.
  • Gene expression can be suppressed by knock out, altering a promoter of a gene, and/or by administering interfering RNAs directed at the factors.
  • a HR enhancer that suppresses non-homologous end-joining can be delivered with plasmid DNA.
  • the plasmid can be a double stranded DNA molecule.
  • the plasmid molecule can also be single stranded DNA.
  • the plasmid can also carry at least one gene.
  • the plasmid can also carry more than one gene.
  • At least one plasmid can also be used.
  • More than one plasmid can also be used.
  • a HR enhancer that suppresses non-homologous end-joining can be delivered with plasmid DNA in conjunction with CRISPR-Cas, primers, and/or a modifier compound.
  • a modifier compound can reduce cellular toxicity of plasmid DNA and improve cellular viability.
  • An HR enhancer and a modifier compound can be introduced to a cell before genomic engineering.
  • the HR enhancer can be a small molecule.
  • the HR enhancer can be delivered to a T cell suspension.
  • An HR enhancer can improve viability of cells transfected with double strand DNA.
  • introduction of double strand DNA can be toxic, FIG. 81 A. and FIG. 81 B.
  • a HR enhancer that suppresses non-homologous end-joining can be delivered with an HR substrate to be integrated.
  • a substrate can be a polynucleic acid.
  • a polynucleic acid can comprise a TCR transgene.
  • a polynucleic acid can be delivered as mRNA (see FIG. 10 and FIG. 14).
  • a polynucleic acid can comprise recombination arms to an endogenous region of the genome for integration of a TCR transgene.
  • a polynucleic acid can be a vector.
  • a vector can be inserted into another vector (e.g., viral vector) in either the sense or anti-sense orientation. Upstream of the 5' LTR region of the viral genome a T7, T3, or other transcriptional start sequence can be placed for in vitro transcription of the viral cassette (see FIG. 3). This vector cassette can be then used as a template for in vitro
  • the single stranded mRNA cassette can be used as a template to generate hundreds to thousands of copies in the form of double stranded DNA (dsDNA) that can be used as a HR substrate for the desired homologous recombination event to integrate a transgene cassette at an intended target site in the genome.
  • dsDNA double stranded DNA
  • This method can circumvent the need for delivery of toxic plasmid DNA for CRISPR mediated homologous recombination.
  • the amount of homologous recombination template available within the cell can be very high. The high amount of homologous recombination template can drive the desired
  • the mRNA can also generate single stranded DNA.
  • Single stranded DNA can also be used as a template for homologous recombination, for example with recombinant AAV (rAAV) gene targeting.
  • mRNA can be reverse transcribed into a DNA
  • homologous recombination HR enhancer in situ This strategy can avoid the toxic delivery of plasmid DNA. Additionally, mRNA can amplify the homologous recombination substrate to a higher level than plasmid DNA and/or can improve the efficiency of homologous recombination.
  • a HR enhancer that suppresses non-homologous end-joining can be delivered as a chemical inhibitor.
  • a HR enhancer can act by interfering with Ligase IV -DNA binding.
  • a HR enhancer can also activate the intrinsic apoptotic pathway.
  • a HR enhancer can also be a peptide mimetic of a Ligase IV inhibitor.
  • a HR enhancer can also be co-expressed with the Cas9 system.
  • a HR enhancer can also be co-expressed with viral proteins, such as E1B55K and/or E4orf6.
  • a HR enhancer can also be SCR7, L755507, or any derivative thereof.
  • a HR enhancer can be delivered with a compound that reduces toxicity of exogenous DNA insertion.
  • mRNAs encoding both the sense and anti-sense strand of the viral vector can be introduced (see FIG. 3).
  • both mRNA strands can be reverse transcribed within the cell and/or naturally anneal to generate dsDNA.
  • the HR enhancer can be delivered to primary cells.
  • a homologous recombination HR enhancer can be delivered by any suitable means.
  • a homologous recombination HR enhancer can also be delivered as an mRNA.
  • a homologous recombination HR enhancer can also be delivered as plasmid DNA.
  • a homologous recombination HR enhancer can also be delivered to immune cells in conjunction with CRISPR-Cas.
  • a homologous recombination HR enhancer can also be delivered to immune cells in conjunction with CRISPR-Cas, a polynucleic acid comprising a TCR sequence, and/or a compound that reduces toxicity of exogenous DNA insertion.
  • a homologous recombination HR enhancer can be delivered to any cells, e.g., to immune cells.
  • a homologous recombination HR enhancer can be delivered to a primary immune cell.
  • a homologous recombination HR enhancer can also be delivered to a T cell, including but not limited to T cell lines and to a primary T cell.
  • a homologous recombination HR enhancer can also be delivered to a CD4+ cell, a CD 8+ cell, and/or a tumor infiltrating cell (TIL).
  • TIL tumor infiltrating cell
  • a homologous recombination HR enhancer can also be delivered to immune cells in conjunction with CRISPR-Cas.
  • a homologous recombination HR enhancer can be used to suppress non-homologous end-joining. In some cases, a homologous recombination HR enhancer can be used to promote homologous directed repair. In some cases, a homologous recombination HR enhancer can be used to promote homologous directed repair after a CRISPR-Cas double stranded break. In some cases, a homologous recombination HR enhancer can be used to promote homologous directed repair after a CRISPR-Cas double stranded break and the knock-in and knock-out of one of more genes. The genes that are knocked-in can be a TCR.
  • the genes that are knocked-out can also be any number of endogenous checkpoint genes.
  • the endogenous checkpoint gene can be selected from the group consisting of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, AAVS SITE (E.G. AAVS1, AAVS2, ETC.), CCR5, HPRT, PPP1R12C, or CISH.
  • the gene can be PD-1.
  • the gene can be an endogenous TCT.
  • the gene can comprise a coding region. In some cases, the gene can comprise a non-coding region.
  • Increase in HR efficiency with an HR enhancer can be or can be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • Decrease in NHEJ with an HR enhancer can be or can be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • Cellular toxicity to exogenous polynucleic acids can be mitigated to improve the engineering of cell, including T cells.
  • cellular toxicity can be reduced by altering a cellular response to polynucleic acid.
  • a polynucleic acid can contact a cell.
  • the polynucleic acids can then be introduced into a cell.
  • a polynucleic acid is utilized to alter a genome of a cell.
  • the cell can die. .
  • insertion of a polynucleic acid can cause apoptosis of a cell as shown in FIG. 18.
  • Toxicity induced by a polynucleic acid can be reduced by using a modifier compound.
  • a modifier compound can disrupt an immune sensing response of a cell.
  • a modifier compound can also reduce cellular apoptosis and pyropoptosis.
  • a modifier compound can be an activator or an inhibitor.
  • the modifier compound can act on any component of the pathways shown in FIG. 19.
  • the modifier compound can act on Caspase-1, TBK1, IRF3, STING, DDX41, DNA-PK, DAI, IFI16, MRE11, cGAS, 2'3'-cGAMP, TREX1, AIM2, ASC, or any combination thereof.
  • the modifier compound can also act on the innate signaling system, thus, it can be an innate signaling modifier.
  • Reducing toxicity to exogenous polynucleic acids can be performed by contacting a compound and a cell.
  • a cell can be pre-treated with a compound prior to contact with a polynucleic acid.
  • a compound and a polynucleic acid are simultaneously introduced to a cell.
  • a compound can be introduced as a cocktail comprising a polynucleic acid, an HR enhancer, and/or CRISPR-Cas.
  • a compound that can be used in the methods and compositions described herein can have one or more of the following characteristics and can have one or more of the function described herein. Despite its one or more functions, a compound described herein can decrease toxicity of exogenous polynucleotides.
  • a compound can modulate a pathway that results in toxicity from exogenously introduced polynucleic acid.
  • a polynucleic acid can be DNA.
  • a polynucleic acid can also be RNA.
  • a polynucleic acid can be single strand.
  • a polynucleic acid can also be double strand.
  • a polynucleic acid can be a vector.
  • a polynucleic acid can also be a naked polynucleic acid.
  • a polynucleic acid can encode for a protein.
  • a polynucleic acid can also have any number of modifications.
  • a polynucleic acid modification can be demethylation, addition of CpG methylation, removal of bacterial methylation, and/or addition of mammalian methylation.
  • a polynucleic acid can also be introduced to a cell as a reagent cocktail comprising additional polynucleic acids, any number of HR enhancers, and/or CRISPR-Cas.
  • a polynucleic acid can also comprise a transgene.
  • a polynucleic acid can comprise a transgene that as a TCR sequence.
  • a compound can also modulate a pathway involved in initiating toxicity to exogenous DNA.
  • a factor can comprise DNA-dependent activator of IFN regulatory factors (DAI), IFN inducible protein 16 (IFI16), DEAD box polypeptide 41 (DDX41), absent in melanoma 2 (AIM2), DNA-dependent protein kinase, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), stimulator of IFN genes (STING), TANK-binding kinase (TBK1), interleukin-1 ⁇ (IL- ⁇ ), MREl l, meiotic recombination 11, Trexl, cysteine protease with aspartate specificity (Caspase-1), three prime repair exonuclease, DNA- dependent activator of IRFs (DAI), IFI16, DDX41, DNA-dependent protein kinase (DNA-PK), meiotic recombination 11 homolog A (MREl 1), and IFN regulatory factor (I), DAI), IFN inducible protein
  • a DNA sensing pathway may generally refer to any cellular signaling pathway that comprises one or more proteins (e.g., DNA sensing proteins) involved in the detection of intracellular nucleic acids, and in some instances, exogenous nucleic acids.
  • a DNA sensing pathway may comprise stimulator of interferon (STING).
  • a DNA sensing pathway may comprise the DNA -dependent activator of IFN-regulatory factor (DAI).
  • Non-limiting examples of a DNA sensing protein include three prime repair exonuclease 1 (TREX1), DEAD-box helicase 41 (DDX41), DNA-dependent activator of IFN-regulatory factor (DAI), Z-DNA -binding protein 1 (ZBP1), interferon gamma inducible protein 16 (IFI16), leucine rich repeat (In FLU) interacting protein 1 (LRRFIP1), DEAH-box helicase 9 (DHX9), DEAH-box helicase 36 (DHX36), Lupus Ku autoantigen protein p70 (Ku70), X-ray repair complementing defective repair in Chinese hamster cells 6 (XRCC6), stimulator of interferon gene (STING), transmembrane protein 173 (TMEM173), tripartite motif containing 32 (TRIM32), tripartite motif containing 56 (TRIM56), ⁇ -catenin
  • TREX1 prime repair exonuclease 1
  • DDX41 DEAD-
  • CARD myeloid differentiation primary response 88
  • AIM2 myeloid differentiation primary response 88
  • ASC apoptosis-associated speck-like protein containing a CARD
  • pro-caspase-1 pro-CASPl
  • caspase-1 caspase-1
  • pro-interleukin 1 beta pro-IL- ⁇
  • pro-interleukin 18 pro-IL-18
  • interleukin 1 beta IL- ⁇
  • interleukin 18 IL-18
  • interferon regulatory factor 1 IRFl
  • IRF3 interferon regulatory Factor 3
  • IRF7 interferon regulatory factor 7
  • ISRE7 interferon-stimulated response element 7
  • ISRE7 interferon-stimulated response element 1/7
  • ISREl/7 nuclear factor kappa B
  • RNA polymerase III RNA polymerase III
  • melanoma differentiation-associated protein 5 MDA-5
  • LGP2 Laboratory of Genetics and Physiology 2
  • LGP2 retinoic acid-induc
  • DAI activates the IRF and NF- ⁇ transcription factors, leading to production of type I interferon and other cytokines.
  • AIM2 upon sensing exogenous intracellular DNA, AIM2 triggers the assembly of the
  • Compounds can also be covalently linked to tags that target a compound for degradation. Besides single modifications, compounds are often modified through a combination of post-translational cleavage and the addition of functional groups through a step-wise mechanism of compound maturation or activation.
  • a compound can reduce production of type I interferons (IFNs), for example, IFN-a, and/or IFN- ⁇ .
  • IFNs type I interferons
  • a compound can also reduce production of proinflammatory cytokines such as tumor necrosis factor-a (TNF-a) and/or interleukin- 1 ⁇ (IL- ⁇ ).
  • TNF-a tumor necrosis factor-a
  • IL- ⁇ interleukin- 1 ⁇
  • a compound can also modulate induction of antiviral genes through the modulation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway.
  • a compound can also modulate transcription factors nuclear factor ⁇ - light-chain enhancer of activated B cells (NF- ⁇ ), and the IFN regulatory factors
  • a compound can also modulate activation of NF- ⁇ , for example modifying phosphorylation of ⁇ by the ⁇ kinase (IKK) complex.
  • a compound can also modulate phosphorylation or prevent phosphorylation of ⁇ .
  • a compound can also modulate activation of IRF3 and/or IRF7.
  • a compound can modulate activation of IRF3 and/or IRF7.
  • a compound can activate TBK1 and/or ⁇ .
  • a compound can also inhibit TBK1 and/or ⁇ .
  • a compound can prevent formation of an enhanceosome complex comprised of IRF3, IRF7, NF- ⁇ and other transcription factors to turn on the transcription of type I IFN genes.
  • a modifying compound can be a TBK1 compound and at least one additional compound, FIG. 88 A and FIG 88. B.
  • a TBK1 compound and a Caspase inhibitor compound can be used to reduce toxicity of double strand DNA, FIG. 89.
  • a compound can prevent cellular apoptosis and/or pyropoptosis.
  • a compound can also prevent activation of an inflammasome.
  • An inflammasome can be an intracellular multiprotein complex that mediates the activation of the proteolytic enzyme caspase- 1 and the maturation of IL- ⁇ .
  • a compound can also modulate AIM2 (absent in melanoma 2).
  • a compound can prevent AIM2 from associating with the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD).
  • a compound can also modulate a homotypic PYD: PYD interaction.
  • a compound can also modulate a homotypic CARD: CARD interaction.
  • a compound can modulate Caspase- 1.
  • a compound can inhibit a process whereby Caspase -1 converts the inactive precursors of IL- ⁇ and IL-18 into mature cytokines.
  • a compound can be a component of a platform to generate a GMP compatible
  • a compound can used to improve cellular therapy.
  • a compound can be used as a reagent.
  • a compound can be combined as a combination therapy.
  • a compound can be utilized ex vivo.
  • a compound can be used for immunotherapy.
  • a compound can be a part of a process that generates a T cell therapy for a patient in need, thereof.
  • a compound is not used to reduce toxicity.
  • a inflammasome In some cases, a inflammasome,
  • RNA PolIII may convert exogenous DNA into RNA for recognition by the RNA sensor RIG-I.
  • the methods of the present disclosure comprise introducing into one or more cells a nucleic acid comprising a first transgene encoding at least one anti-DNA sensing protein.
  • An anti-DNA sensing protein may generally refer to any protein that alters the activity or expression level of a protein corresponding to a DNA sensing pathway (e.g., a DNA sensing protein).
  • an anti-DNA sensing protein may degrade (e.g., reduce overall protein level) of one or more DNA sensing proteins.
  • an anti-DNA sensing protein may fully inhibit one or more DNA sensing proteins.
  • an anti-DNA sensing protein may partially inhibit one or more DNA sensing proteins.
  • an anti-DNA sensing protein may inhibit the activity of at least one DNA sensing protein by at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%.
  • an anti-DNA sensing protein may decrease the amount of at least one DNA sensing protein by at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%.
  • Cell viability may be increased by introducing viral proteins during a genomic engineering procedure, which can inhibit the cells ability to detect exogenous DNA.
  • an anti-DNA sensing protein may promote the translation (e.g., increase overall protein level) of one or more DNA sensing proteins.
  • an anti-DNA sensing protein may protect or increase the activity of one or more DNA sensing proteins.
  • an anti-DNA sensing protein may increase the activity of at least one DNA sensing protein by at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%.
  • an anti-DNA sensing protein may increase the amount of at least one DNA sensing protein by at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%.
  • an anti-DNA sensing inhibitor may be a competitive inhibitor or activator of one or more DNA sensing proteins.
  • an anti-DNA sensing protein may be a non-competitive inhibitor or activator of a DNA sensing protein.
  • an anti-DNA sensing protein may also be a DNA sensing protein (e.g., TREX1).
  • Non-limiting examples of anti-DNA sensing proteins include cellular FLICE- inhibitory protein (c-FLiP), Human cytomegalovirus tegument protein (HCMV pUL83), dengue virus specific NS2B-NS3 (DENV NS2B-NS3), Protein E7-Human papillomavirus type 18 (HPV18 E7), hAd5 El A, Herpes simplex virus immediate-early protein ICP0 (HSV1 ICP0), Vaccinia virus B 13
  • VACV B 13 Vaccinia virus C16
  • VACV CI 6 three prime repair exonuclease 1
  • TREX1 three prime repair exonuclease 1
  • HoV-NL63 human coronavirus NL63
  • SARS-CoV severe actute respiratory syndrome coronavirus
  • HBV Pol hepatitis B virus DNA polymerase
  • PEDV porcine epidemic diarrhea virus
  • ADAR1 adenosine deaminase
  • E3L E3L
  • p202 a phosphorylated form of a protein thereof, and any combination or derivative thereof.
  • HCMV pUL83 may disrupt a DNA sensing pathway by inhibiting activation of the STING-TBK1-IRF3 pathway by interacting with the pyrin domain on IFI16 (e.g., nuclear IFI16) and blocking its oligomerization and subsequent downstream activation.
  • DENV Ns2B-NS3 may disrupt a DNA sensing pathway by degrading STING.
  • HPV18 E7 may disrupt a DNA sensing pathway by blocking the cGAS/STING pathway signaling by binding to STING.
  • hAd5 El A may disrupt a DNA sensing pathway by blocking the cGAS/STING pathway signaling by binding to STING.
  • FIG 104 A and FIG 104B show cells transfected with a CRISPR system, an exogenous polynucleic acid, and an hAd5 El A (El A) or HPV18 E7 protein.
  • HSV1 ICPO may disrupt a DNA sensing pathway by degradation of IFI16 and/or delaying recruitment of IFI16 to the viral genome.
  • VACV B 13 may disrupt a DNA sensing pathway by blocking Caspase 1 -dependant inflammasome activation and Caspase 8- dependent extrinsic apoptosis.
  • VACV C16 may disrupt a DNA sensing pathway by blocking innate immune responses to DNA, leading to decreased cytokine expression.
  • a compound can be an inhibitor.
  • a compound can also be an activator.
  • a compound can be any compound.
  • a compound can also be combined with at least one compound.
  • one or more compounds can behave synergistically. For example, one or more compounds can reduce cellular toxicity when introduced to a cell at once as shown in FIG. 20.
  • a compound can be Pan Caspase Inhibitor Z-VAD-FMK and/or Z-VAD-FMK.
  • a compound can be a derivative of any number of known compounds that modulate a pathway involved in initiating toxicity to exogenous DNA.
  • a compound can also be modified.
  • a compound can be modified by any number of means, for example, a modification to a compound can comprise deuteration, lipidization, glycosylation, alkylation, PEGylation, oxidation, phosphorylation, sulfation, amidation, biotinylation, citrullination, isomerization, ubiquitylation, protonation, small molecule conjugations, reduction, dephosphorylation, nitrosylation, and/or proteolysis.
  • a modification can also be post- translational.
  • a modification can be pre-translation.
  • a modification can occur at distinct amino acid side chains or peptide linkages and can be mediated by enzymatic activity.
  • a modification can occur at any step in the synthesis of a compound.
  • polynucleic acid can be modified to also reduce toxicity.
  • a polynucleic acid can be modified to reduce detection of a polynucleic acid, e.g., an exogenous polynucleic acid.
  • a polynucleic acid can also be modified to reduce cellular toxicity.
  • a polynucleic acid can be modified by one or more of the methods depicted in FIG. 21.
  • a polynucleic acid can also be modified in vitro or in vivo.
  • a compound or modifier compound can reduce cellular toxicity of plasmid DNA by or by about 10%
  • Unmethylated polynucleic acid can also reduce toxicity.
  • an unmethylated polynucleic acid comprising at least one engineered antigen receptor flanked by at least two recombination arms complementary to at least one genomic region can be used to reduce cellular toxicity.
  • the polynucleic acid can also be naked polynucleic acids.
  • the polynucleic acids can also have mammalian methylation, which in some cases will reduce toxicity as well.
  • a polynucleic acid can also be modified so that bacterial methylation is removed and mammalian methylation is introduced. Any of the modifications described herein can apply to any of the polynucleic acids as described herein.
  • Polynucleic acid modifications can comprise demethylation, addition of CpG methylation, removal of bacterial methylation, and/or addition of mammalian methylation.
  • a modification can be converting a double strand polynucleic acid into a single strand polynucleic acid.
  • a single strand polynucleic acid can also be converted into a double strand polynucleic acid.
  • a polynucleic acid can be methylated (e.g. Human methylation) to reduce cellular toxicity.
  • methylated e.g. Human methylation
  • modified polynucleic acid can comprise a TCR sequence or chimeric antigen receptor (CAR).
  • the polynucleic acid can also comprise an engineered extracellular receptor.
  • Mammalian methylated polynucleic acid comprising at least one engineered antigen receptor can be used to reduce cellular toxicity.
  • a polynucleic acid can be modified to comprise mammalian methylation.
  • a polynucleic acid can be methylated with mammalian methylation so that it is not recognized as foreign by a cell.
  • Polynucleic acid modifications can also be performed as part of a culturing process.
  • Demethylated polynucleic acid can be produced with genomically modified bacterial cultures that do not introduce bacterial methylation. These polynucleic acids can later be modified to contain mammalian methylation, e.g., human methylation.
  • Toxicity can also be reduced by introducing viral proteins during a genomic engineering procedure.
  • viral proteins can be used to block DNA sensing and reduce toxicity of a donor nucleic acid encoding for an exogenous TCR or CRISPR system.
  • An evasion strategy employed by a virus to block DNA sensing can be sequestration or modification of a viral nucleic acid; interference with specific post-translational modifications of PRRs or their adaptor proteins; degradation or cleavage of pattern recognition receptors (PRRs) or their adaptor proteins; sequestration or relocalization of PRRs, or any combination thereof.
  • a viral protein may be introduced that can block DNA sensing by any of the evasion strategies employed by a virus.
  • a viral protein can be or can be derived from a virus such as Human cytomegalovirus (HCMV), Dengue virus (DENV), Human Papillomavirus Virus (HPV), Herpes Simplex Virus type 1 (HSV1), Vaccinia Virus (VACV), Human coronaviruses (HCoVs), Severe acute respiratory syndrome (SARS) corona virus (SARS-Cov), Hepatitis B virus, Porcine epidemic diarrhea virus, or any combination thereof.
  • An introduced viral protein can prevent RIG-I-like receptors (RLRs) from accessing viral RNA by inducing formation of specific replication compartments that can be confined by cellular membranes, or in other cases to replicate on organelles, such as an endoplasmic reticulum, a Golgi apparatus, mitochondria, or any combination thereof.
  • RLRs RIG-I-like receptors
  • a virus of the invention can have
  • an RLR signaling pathway can be inhibited.
  • a Lys63-linked ubiquitylation of RIG-I can be inhibited or blocked to prevent activation of RIG-I signaling.
  • a viral protein can target a cellular E3 ubiquitin ligase that can be responsible for ubiquitylation of RIG-I.
  • a viral protein can also remove a ubiquitylation of RIG-I.
  • viruses can inhibit a ubiquitylation (e.g., Lys63 -linked) of RIG-I independent of protein-protein interactions, by modulating the abundance of cellular microRNAs or through RNA-protein interactions.
  • viral proteins can process a 5 '-triphosphate moiety in the viral RNA, or viral nucleases can digest free double-stranded RNA (dsRNA). Furthermore, viral proteins, can bind to viral RNA to inhibit the recognition of pathogen-associated molecular patterns (PAMPs) by RIG-I. Some viral proteins can manipulate specific post-translational modifications of RIG-I and/or MDA5, thereby blocking their signaling abilities. For example, viruses can prevent the Lys63-linked ubiquitylation of RIG-I by encoding viral deubiquitylating enzymes (DUBs). In other cases, a viral protein can antagonize a cellular E3 ubiquitin ligase, tripartite motif protein 25
  • TAM25 and/or Riplet, thereby also inhibiting RIG-I ubiquitylation and thus its activation.
  • a viral protein can bind to TRIM25 to block sustained RIG-I signaling.
  • a viral protein can prevent a PP la-mediated or ⁇ -mediated dephosphorylation of MDA5, keeping it in its phosphorylated inactive state.
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • PACT protein kinase R activator
  • An NS3 protein from DENV virus can target the trafficking factor 14-3-3 ⁇ to prevent translocation of RIG-I to MAVS at the mitochondria.
  • a viral protein can cleave RIG-I, MDA5 and/or MAVS.
  • viral proteins can be introduced to subvert cellular degradation pathways to inhibit RLR-MAVS-dependent signaling.
  • an X protein from hepatitis B virus (HBV) and the 9b protein from severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) can promote the ubiquitylation and degradation of MAVS.
  • an introduced viral protein can allow for immune evasion of cGAS, IFI16, STING, or any combination thereof.
  • a viral protein can use the cellular 3 '-repair exonuclease 1 (TREX1) to degrade excess reverse transcribed viral DNA.
  • TREX1 cellular 3 '-repair exonuclease 1
  • the a viral capsid can recruit host-encoded factors, such as cyclophilin A (CYPA), which can prevent the sensing of reverse transcribed DNA by cGAS.
  • CYPA cyclophilin A
  • an introduced viral protein can bind to both viral DNA and cGAS to inhibit the activity of cGAS.
  • STING stimulator of interferon
  • Pol polymerase
  • HBV hepatitis B virus
  • PBPs papain-like proteases
  • HCV-NL63 human coronavirus NL63
  • SARS-CoV severe acute respiratory syndrome
  • An introduced viral protein can also bind to STING and inhibit its activation or cleave STING to inactivate it.
  • IFI16 can be inactivated.
  • a viral protein can target IFI16 for proteasomal degradation or bind to IFI16 to prevent its oligomerization and thus its activation.
  • a viral protein to be introduced can be or can be derived from: HCMV pUL83, DENV NS2B-NS3, HPV18 E7, hAd5 E1A, HSV1 ICP0, VACV B 13, VACV C16, TREX1, HCoV-NL63, SARS-Cov, HBV Pol PEDV, or any combination thereof.
  • a viral protein can be adenoviral.
  • Adenoviral proteins can be adenovirus 4 E1B55K, E4orf6 protein.
  • a viral protein can be a B 13 vaccine virus protein.
  • Viral proteins that are introduced can inhibit cytosolic DNA recognition, sensing, or a combination.
  • a RIP pathway can be inhibited.
  • a cellular FLICE (FADD-like IL- lbeta-converting enzyme)-inhibitory protein (c-FLIP) pathway can be introduced to a cell.
  • c-FLIP can be expressed as long (c-FLIPL), short (c -FLIPS), and c-FLIPR splice variants in human cells, c- FLIP can be expressed as a splice variant.
  • c-FLIP can also be known as Casper, iFLICE, FLAME-1, CASH, CLARP, MRIT, or usurpin.
  • c-FLIP can bind to FADD and/or caspase-8 or -10 and TRAIL receptor 5 (DR5). This interaction in turn prevents Death-Inducing Signaling Complex (DISC) formation and subsequent activation of the caspase cascade.
  • c-FLIPL and c-FLIPS are also known to have multifunctional roles in various signaling pathways, as well as activating and/or upregulating several cytoprotective and pro-survival signaling proteins including Akt, ERK, and NF- ⁇ . In some cases, c-FLIP can be introduced to a cell to increase viability.
  • STING can be inhibited.
  • a caspase pathway is inhibited.
  • a DNA sensing pathway can be a cytokine-based inflammatory pathway and/or an interferon alpha expressing pathway.
  • a multimodal approach is taken where at least one DNA sensing pathway inhibitor is introduced to a cell.
  • an inhibitor of DNA sensing can reduce cell death and allow for improved integration of an exogenous TCR transgene.
  • a multimodal approach can be a STING and Caspase inhibitor in combination with a TBK inhibitor.
  • An introduced viral protein can reduce cellular toxicity of plasmid DNA by or by about 10%, 20%,
  • a viral protein can improve cellular viability by or by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • gRNA can be used to reduce toxicity.
  • a gRNA can be engineered to bind within a filler region of a vector.
  • a vector can be a minicircle DNA vector.
  • a minicircle vector can be used in conjunction with a viral protein.
  • a minicircle vector can be used in conjunction with a viral protein and at least one additional toxicity reducing agent.
  • genomic disruptions can be performed more efficiently.
  • an enzyme can be used to reduce DNA toxicity.
  • an enzyme such as Dpnl can be utilized to remove methylated targets on a DNA vector or transgene.
  • a vector or transgene can be pre-treated with Dpnl prior to electroporation.
  • Type IIM restriction endonucleases such as Dpnl, are able to recognize and cut methylated DNA.
  • a minicircle DNA is treated with Dpnl.
  • Naturally occurring restriction endonucleases are categorized into four groups (Types I, II III, and IV).
  • a restriction endonuclease such as Dpnl or a CRISPR system endonuclease is utilized to prepare engineered cells.
  • an adenoviral protein or functional portion thereof introducing at least one polynucleic acid encoding at least one exogenous receptor sequence; and genomically disrupting at least one genome with at least one endonuclease or portion thereof.
  • an adenoviral protein or function portion thereof is E1B55K, E4orf6, Scr7, L755507, NS2B3, HPV18 E7, hAd5 E1A, or a combination thereof.
  • An adenoviral protein can be selected from a serotype 1 to 57. In some cases, an adenoviral protein serotype is serotype 5.
  • an engineered adenoviral protein or portion thereof has at least one modification.
  • a modification can be a substitution, insertion, deletion, or modification of a sequence of said adenoviral protein.
  • a modification can be an insertion.
  • An insertion can be a AGIPA insertion.
  • a modification is a substitution.
  • a substitution can be a H to A at amino acid position 373 of a protein sequence.
  • a polynucleic acid can be DNA or RNA.
  • a polynucleic acid can be DNA.
  • DNA can be minicircle DNA.
  • an exogenous receptor sequence can be selected from the group consisting of a sequence of a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), and any portion or derivative thereof.
  • An exogenous receptor sequence can be a TCR sequence.
  • An endonuclease can be selected from the group consisting of CRISPR, TALEN, transposon-based, ZEN, meganuclease, Mega-TAL, and any portion or derivative thereof.
  • An endonuclease can be CRISPR.
  • CRISPR can comprise at least one Cas protein.
  • a Cas protein can be selected from the group consisting of Casl, Cas IB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl , Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Cpfl, c2cl, c2c3, Cas9HiFi, homologues thereof or modified versions thereof.
  • a Cas protein can be Cas9.
  • CRISPR creates a double strand break in a genome.
  • a genome can comprise at least one gene.
  • an exogenous receptor sequence is introduced into at least one gene.
  • An introduction can disrupt at least one gene.
  • a gene can be CISH, PD-1, TRA, TRB, or a combination thereof.
  • a cell can be human.
  • a human cell can be immune.
  • An immune cell can be CD3+, CD4+,
  • a method can further comprise expanding a cell.
  • a method of making an engineered cell comprising: virally introducing at least one polynucleic acid encoding at least one exogenous T cell receptor (TCR) sequence; and genomically disrupting at least one gene with at least one endonuclease or functional portion thereof.
  • a virus can be selected from retrovirus, lentivirus, adenovirus, adeno-associated virus, or any derivative thereof.
  • a virus can be an adeno-associated virus (AAV).
  • An AAV can be serotype 5.
  • An AAV can comprise at least one modification.
  • a modification can be a chemical modificaiton.
  • a polynucleic acid can be DNA, RNA, or any modification thereof.
  • a polynucleic acid can be DNA.
  • DNA is minicircle DNA.
  • a polynucleic acid can further comprise at least one homology arm flanking a TCR sequence.
  • a homology arm can comprise a complementary sequence at least one gene.
  • a gene can be an endogenous gene.
  • An endogenous gene can be a checkpoint gene.
  • a method can further comprise at least one toxicity reducing agent.
  • a toxicity reducing agent can be a viral protein or an inhibitor of the cytosolic DNA sensing pathway.
  • a viral protein can be E 1B55K, E4orf6, Scr7, L755507, NS2B3, HPV18 E7, hAd5 E l A, or a combination thereof
  • a method can further comprise expansion of cells.
  • an inhibitor of the cytosolic DNA sensing pathway can be used.
  • An inhibitor of the cytosolic DNA sensing pathway can be cellular FLICE (FADD-like IL- i -converting enzyme) -inhibitory protein (c-FLIP).
  • Cell viability and/or the efficiency of integration of a transgene into a genome of one or more cells may be measured using any method known in the art. In some cases, cell viability and/or efficiency of integration may be measured using trypan blue exclusion, terminal deoxynucleotidyl
  • TUNEL transferase dUTP nick end labeling
  • FACS fluorescence -activated cell sorting
  • real-time PCR or droplet digital PCR.
  • FACS fluorescence -activated cell sorting
  • apoptosis of may be measured using
  • polynucleotides and compositions comprising the proteins and/or polynucleotides described herein can be delivered to a target cell by any suitable means.
  • Suitable cells can include but are not limited to eukaryotic and prokaryotic cells and/or cell lines.
  • Non-limiting examples of such cells or cell lines generated from such cells include COS, CHO (e.g. ,
  • HEK293 e.g., HEK293-F, HEK293-H,
  • the cell line is a CHO-K1
  • suitable primary cells include peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), and other blood cell subsets such as, but not limited to, T cell, a natural killer cell, a monocyte, a natural killer T cell, a monocyte- precursor cell, a hematopoietic stem cell or a non-pluripotent stem cell.
  • the cell can be any immune cells including any T-cell such as tumor infiltrating cells (TILs), such as CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, or any other type of T-cell.
  • TILs tumor infiltrating cells
  • the T cell can also include memory T cells, memory stem T cells, or effector T cells.
  • the T cells can also be selected from a bulk population, for example, selecting T cells from whole blood.
  • the T cells can also be expanded from a bulk population.
  • the T cells can also be skewed towards particular populations and phenotypes.
  • the T cells can be skewed to phenotypically comprise, CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and/or IL-7Ra(+).
  • Suitable cells can be selected that comprise one of more markers selected from a list comprising: CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and/or IL-7Ra(+).
  • Suitable cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells and mesenchymal stem cells.
  • stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells and mesenchymal stem cells.
  • Suitable cells can comprise any number of primary cells, such as human cells, non-human cells, and/or mouse cells.
  • Suitable cells can be progenitor cells.
  • Suitable cells can be derived from the subject to be treated (e.g., patient). Suitable cells can be derived from a human donor.
  • Suitable cells can be stem memory T S CM cells comprised of CD45RO (-), CCR7(+), CD45RA (+), CD62L+ (L-selectin), CD27+, CD28+ and IL-7Ra+, stem memory cells can also express CD95, IL-2R , CXCR3, and LFA-1, and show numerous functional attributes distinctive of stem memory cells.
  • Suitable cells can be central memory T C M cells comprising L-selectin and CCR7, central memory cells can secrete, for example, IL-2, but not IFNy or IL-4.
  • Suitable cells can also be effector memory T EM cells comprising L-selectin or CCR7 and produce, for example, effector cytokines such as IFNy and IL-4.
  • a method of attaining suitable cells can comprise selecting cells.
  • a cell can comprise a marker that can be selected for the cell.
  • marker can comprise GFP, a resistance gene, a cell surface marker, an endogenous tag.
  • Cells can be selected using any endogenous marker.
  • Suitable cells can be selected using any technology. Such technology can comprise flow cytometry and/or magnetic columns. The selected cells can then be infused into a subject. The selected cells can also be expanded to large numbers. The selected cells can be expanded prior to infusion.
  • transcription factors and nucleases as described herein can be delivered using vectors, for example
  • Transgenes encoding polynucleotides can be similarly delivered.
  • Any vector systems can be used including, but not limited to, plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors;
  • herpesvirus vectors and adeno-associated virus vectors, etc.
  • any of these vectors can comprise one or more transcription factor, nuclease, and/or transgene.
  • CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega-TAL molecules and/or transgenes are introduced into the cell, CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega-TAL molecules and/or transgenes can be carried on the same vector or on different vectors. When multiple vectors are used, each vector can comprise a sequence encoding one or multiple CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega-TAL molecules and/or transgenes.
  • Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding engineered CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega-TAL molecules and/or transgenes in cells (e.g., mammalian cells) and target tissues. Such methods can also be used to administer nucleic acids encoding CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega-TAL molecules and/or transgenes to cells in vitro.
  • nucleic acids encoding CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega-TAL molecules and/or transgenes can be administered for in vivo or ex vivo immunotherapy uses.
  • Non-viral vector delivery systems can include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • Viral vector delivery systems can include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Methods of non-viral delivery of nucleic acids include electroporation, lipofection, nucleofection, gold nanoparticle delivery, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, mRNA, artificial virions, and agent- enhanced uptake of DNA. Sonoporation using, e.g. , the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids.
  • Additional exemplary nucleic acid delivery systems include those provided by AMAXA ® Biosystems (Cologne, Germany), Life Technologies (Frederick, Md.), MAXCYTE, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc. (see for example U.S. Pat. No. 6,008,336).
  • Lipofection reagents are sold commercially (e.g., TRANSFECTAM ® and LIPOFECTIN ® ). Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration). Additional methods of delivery include the use of packaging the nucleic acids to be delivered into EnGeneIC delivery vehicles (EDVs).
  • EDVs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV.
  • the antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis.
  • Vectors including viral and non-viral vectors containing nucleic acids encoding engineered CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega-TAL molecules, transposon and/or transgenes can also be administered directly to an organism for transduction of cells in vivo.
  • naked DNA or mRNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. More than one route can be used to administer a particular composition. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.
  • a vector encoding for an exogenous TCR can be shuttled to a cellular nuclease.
  • a vector can contain a nuclear localization sequence (NLS).
  • a vector can also be shuttled by a protein or protein complex.
  • Cas9 can be used as a means to shuttle a minicircle vector.
  • Cas can comprise a NLS.
  • a vector can be pre-complexed with a Cas protein prior to electroporation.
  • a Cas protein that can be used for shuttling can be a nuclease-deficient Cas9 (dCas9) protein.
  • a Cas protein that can be used for shuttling can be a nuclease-competent Cas9.
  • Cas protein can be pre-mixed with a guide RNA and a plasmid encoding an exogenous TCR.
  • vectors that can be used include, but not limited to, Bacterial: pBs, pQE-9 (Qiagen), phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5
  • any other plasmids and vectors can be used as long as they are replicable and viable in a selected host.
  • Any vector and those commercially available (and variants or derivatives thereof) can be engineered to include one or more recombination sites for use in the methods.
  • Such vectors can be obtained from, for example, Vector Laboratories Inc., Invitrogen,
  • vectors of interest include eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-Cl, pPUR, pMAM, pMAMneo, pBHOl, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCHl 10, and pKK232-8
  • vectors include pUC18, pUC19, pBlueScript, pSPORT, cosmids, phagemids, YAC's (yeast artificial chromosomes), BAC's (bacterial artificial chromosomes), PI (Escherichia coli phage), pQE70, pQE60, pQE9 (quagan), pBS vectors, PhageScript vectors, BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), pcDNA3 (Invitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORTl, pSPORT2,
  • Additional vectors of interest can also include pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBa- cHis2, pcDNA3.1/His, pcDNA3.1(-)/Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pA081S, pPICZ, pPICZA, pPICZB, pPICZC, pGAPZA, pGAPZB, pGAPZC, pBlue-Bac4.5, P BlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, pIND(SPl), pVgRXR, pcDNA
  • pcDNAl l/Amp, pcDNA3.1, pcDNA3.1/Zeo, pSe, SV2, pRc/CMV2, pRc/ RSV, pREP4, pREP7, pREP8, pREP9, pREP 10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, and pCRBac from Invitrogen;
  • X ExCell X gtl 1, pTrc99A, pKK223-3, pGEX- ⁇ T, pGEX-2T, pGEX-2TK, pGEX-4T- 1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-l, pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL
  • vectors can be used to express a gene, e.g., a transgene, or portion of a gene of interest.
  • a gene of portion or a gene can be inserted by using any method
  • a method can be a restriction enzyme-based technique.
  • Vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g. , intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g. , lymphocytes, T cells, bone marrow aspirates, tissue biopsy), followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector. Prior to or after selection, the cells can be expanded.
  • a vector can be a minicircle vector, FIG. 43.
  • a cell can be transfected with a minicircle vector and a CRISPR system.
  • a minicircle vector concentration can be from 0.5 nanograms to 50 micrograms.
  • the amount of nucleic acid e.g., ssDNA, dsDNA, RNA
  • the amount of nucleic acid may be varied to optimize transfection efficiency and/or cell viability.
  • less than about 100 picograms of nucleic acid may be added to each cell sample (e.g., one or more cells being
  • micrograms at least about 6 micrograms, at least about 6.5 micrograms, at least about 7 micrograms, at least about 7.5 micrograms, at least about 8 micrograms, at least about 8.5 micrograms, at least about 9 micrograms, at least about 9.5 micrograms, at least about 10 micrograms, at least about 11 micrograms, at least about 12 micrograms, at least about 13 micrograms, at least about 14
  • micrograms at least about 15 micrograms, at least about 20 micrograms, at least about 25
  • micrograms at least about 30 micrograms, at least about 35 micrograms, at least about 40
  • micrograms, at least about 45 micrograms, or at least about 50 micrograms, of nucleic acid may be added to each cell sample (e.g., one or more cells being electroporated). For example, 1 microgram of dsDNA may be added to each cell sample for electroporation.
  • the amount of nucleic acid (e.g., dsDNA) required for optimal transfection efficiency and/or cell viability may be specific to the cell type.
  • the amount of nucleic acid (e.g., dsDNA) used for each sample may directly correspond to the transfection efficiency and/or cell viability.For example, a range of concentrations of minicircle transfections are shown in FIG. 70 A, FIG. 70 B, and FIG. 73.
  • FIG. 74 A representative flow cytometry experiment depicting a summary of efficiency of integration of a minicircle vector transfected at a 5 and 20 microgram concentration is shown in FIG. 74, FIG. 78, and FIG. 79.
  • a transgene encoded by a minicircle vector can integrate into a cellular genome. In some cases, integration of a transgene encoded by a minicircle vector is in the forward direction, FIG. 75. In other cases, integration of a transgene encoded by a minicircle vector is in the reverse direction.
  • the transfection efficiency of cells with any of the nucleic acid delivery platforms described herein, for example, nucleofection or electroporation can be or can be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more than 99.9%.
  • Electroporation parameters may be adjusted to optimize transfection efficiency and/or cell viability. Electroporation devices can have multiple electrical wave form pulse settings such as exponential decay, time constant and square wave. Every cell type has a unique optimal Field Strength (E) that is dependent on the pulse parameters applied (e.g., voltage, capacitance and resistance). Application of optimal field strength causes electropermeabilization through induction of transmembrane voltage, which allows nucleic acids to pass through the cell membrane. In some cases, the electroporation pulse voltage, the electroporation pulse width, number of pulses, cell density, and tip type may be adjusted to optimize transfection efficiency and/or cell viability.
  • E Field Strength
  • electroporation pulse voltage may be varied to optimize transfection efficiency and/or cell viability.
  • the electroporation voltage may be less than about 500 volts.
  • the electroporation voltage may be at least about 500 volts, at least about 600 volts, at least about 700 volts, at least about 800 volts, at least about 900 volts, at least about 1000 volts, at least about 1 100 volts, at least about 1200 volts, at least about 1300 volts, at least about 1400 volts, at least about 1500 volts, at least about 1600 volts, at least about 1700 volts, at least about 1800 volts, at least about 1900 volts, at least about 2000 volts, at least about 2100 volts, at least about 2200 volts, at least about 2300 volts, at least about 2400 volts, at least about 2500 volts, at least about 2600 volts, at least about 2700
  • the electroporation pulse voltage required for optimal transfection efficiency and/or cell viability may be specific to the cell type. For example, an electroporation voltage of 1900 volts may optimal (e.g., provide the highest viability and/or transfection efficiency) for macrophage cells. In another example, an electroporation voltage of about 1350 volts may optimal (e.g., provide the highest viability and/or transfection efficiency) for Jurkat cells or primary human cells such as T cells. In some cases, a range of electroporation voltages may be optimal for a given cell type. For example, an electroporation voltage between about 1000 volts and about 1300 volts may optimal (e.g., provide the highest viability and/or transfection efficiency) for human 578T cells.
  • electroporation pulse width may be varied to optimize transfection efficiency and/or cell viability.
  • the electroporation pulse width may be less than about 5 milliseconds.
  • the electroporation width may be at least about 5 milliseconds, at least about 6 milliseconds, at least about 7 milliseconds, at least about 8 milliseconds, at least about 9 milliseconds, at least about 10 milliseconds, at least about 1 1 milliseconds, at least about 12 milliseconds, at least about 13 milliseconds, at least about 14 milliseconds, at least about 15 milliseconds, at least about 16 milliseconds, at least about 17 milliseconds, at least about 18 milliseconds, at least about 19 milliseconds, at least about 20 milliseconds, at least about 21 milliseconds, at least about 22 milliseconds, at least about 23 milliseconds, at least about 24 milliseconds, at least about 25 milliseconds, at least about
  • the electroporation pulse width required for optimal transfection efficiency and/or cell viability may be specific to the cell type. For example, an electroporation pulse width of 30 milliseconds may optimal (e.g., provide the highest viability and/or transfection efficiency) for macrophage cells. In another example, an electroporation width of about 10 milliseconds may optimal (e.g., provide the highest viability and/or transfection efficiency) for Jurkat cells. In some cases, a range of electroporation widths may be optimal for a given cell type. For example, an electroporation width between about 20 milliseconds and about 30 milliseconds may optimal (e.g., provide the highest viability and/or transfection efficiency) for human 578T cells.
  • the number of electroporation pulses may be varied to optimize transfection efficiency and/or cell viability.
  • electroporation may comprise a single pulse.
  • electroporation may comprise more than one pulse.
  • electroporation may comprise 2 pulses, 3 pulses, 4 pulses, 5 pulses 6 pulses, 7 pulses, 8 pulses, 9 pulses, or 10 or more pulses.
  • the number of electroporation pulses required for optimal transfection efficiency and/or cell viability may be specific to the cell type. For example, electroporation with a single pulse may be optimal (e.g., provide the highest viability and/or transfection efficiency) for macrophage cells.
  • electroporation with a 3 pulses may be optimal (e.g., provide the highest viability and/or transfection efficiency) for primary cells.
  • a range of electroporation widths may be optimal for a given cell type.
  • electroporation with between about 1 to about 3 pulses may be optimal (e.g., provide the highest viability and/or transfection efficiency) for human cells.
  • the starting cell density for electroporation may be varied to optimize transfection efficiency and/or cell viability. In some cases, the starting cell density for electroporation may be less than about lxlO 5 cells. In some cases, the starting cell density for electroporation may be at least about lxlO 5 cells, at least about 2xl0 5 cells, at least about 3xl0 5 cells, at least about 4xl0 5 cells, at least about 5xl0 5 cells, at least about 6xl0 5 cells, at least about 7xl0 5 cells, at least about 8xl0 5 cells, at least about 9xl0 5 cells, at least about lxlO 6 cells, at least about 1.5xl0 6 cells, at least about 2xl0 6 cells, at least about 2.5xl0 6 cells, at least about 3xl0 6 cells, at least about 3.5xl0 6 cells, at least about 4xl0 6 cells, at least about 4.5xl0 6 cells, at least about 5xl0
  • 6xl0 6 cells at least about 6.5xl0 6 cells, at least about 7xl0 6 cells, at least about 7.5xl0 6 cells, at least about 8xl0 6 cells, at least about 8.5xl0 6 cells, at least about 9xl0 6 cells, at least about 9.5xl0 6 cells, at least about lxlO 7 cells, at least about 1.2xl0 7 cells, at least about 1.4xl0 7 cells, at least about 1.6xl0 7 cells, at least about 1.8xl0 7 cells, at least about 2xl0 7 cells, at least about 2.2xl0 7 cells, at least about
  • the starting cell density for electroporation required for optimal transfection efficiency and/or cell viability may be specific to the cell type.
  • a starting cell density for electroporation of 1.5xl0 6 cells may optimal (e.g., provide the highest viability and/or transfection efficiency) for macrophage cells.
  • a starting cell density for electroporation of 5xl0 6 cells may optimal (e.g., provide the highest viability and/or transfection efficiency) for human cells.
  • a range of starting cell densities for electroporation may be optimal for a given cell type. For example, a starting cell density for electroporation between of 5.6xl0 6 and 5 xlO 7 cells may optimal (e.g., provide the highest viability and/or transfection efficiency) for human cells such as T cells.
  • the efficiency of integration of a nucleic acid sequence encoding an exogenous TCR into a genome of a cell with, for example, a CRISPR system can be or can be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more than 99.9%.
  • Integration of an exogenous polynucleic acid can be measured using any technique.
  • integration can be measured by flow cytometry, surveyor nuclease assay (FIG. 56), tracking of indels by decomposition (TIDE), FIG. 71 and FIG. 72, junction PCR, or any combination thereof.
  • a representative TIDE analysis is shown for percent gene editing efficiency as show for PD-1 and CTLA-4 guide RNAs, FIG. 35 and FIG. 36.
  • a representative TIDE analysis for CISH guide RNAs is shown from FIG. 62 to FIG. 67 A and B.
  • transgene integration can be measured by PCR, FIG. 77, FIG. 80, and FIG. 95.
  • Ex vivo cell transfection can also be used for diagnostics, research, or for gene therapy (e.g., via re- infusion of the transfected cells into the host organism).
  • cells are isolated from the subject organism, transfected with a nucleic acid (e.g., gene or cDNA), and re-infused back into the subject organism (e.g., patient).
  • a nucleic acid e.g., gene or cDNA
  • the amount of cells that are necessary to be therapeutically effective in a patient may vary depending on the viability of the cells, and the efficiency with which the cells have been genetically modified
  • the product (e.g., multiplication) of the viability of cells post genetic modification and the efficiency of integration of a transgene may correspond to the therapeutic aliquot of cells available for administration to a subject.
  • an increase in the viability of cells post genetic modification may correspond to a decrease in the amount of cells that are necessary for administration to be therapeutically effective in a patient.
  • an increase in the efficiency with which a transgene has been integrated into one or more cells may correspond to a decrease in the amount of cells that are necessary for administration to be therapeutically effective in a patient.
  • determining an amount of cells that are necessary to be therapeutically effective may comprise determining a function corresponding to a change in the viability of cells over time. In some cases, determining an amount of cells that are necessary to be therapeutically effective may comprise determining a function corresponding to a change in the efficiency with which a transgene may be integrated into one or more cells with respect to time dependent variables (e.g., cell culture time, electroporation time, cell stimulation time),
  • Cells before, after, and/or during transplantation can be functional.
  • transplanted cells can be functional for at least or at least about 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, 6, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 days after transplantation.
  • Transplanted cells can be functional for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after transplantation.
  • Transplanted cells can be functional for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 years after transplantation.
  • transplanted cells can be functional for up to the lifetime of a recipient.
  • transplanted cells can function at 100% of its normal intended operation.
  • Transplanted cells can also function 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of its normal intended operation.
  • Transplanted cells can also function over 100% of its normal intended operation. For example,
  • transplanted cells can function 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 or more % of its normal intended operation.
  • compositions described throughout can be formulation into a pharmaceutical medicament and be used to treat a human or mammal, in need thereof, diagnosed with a disease, e.g. , cancer.
  • These medicaments can be co-administered with one or more T cells (e.g., engineered T cells) to a human or mammal, together with one or more chemotherapeutic agent or chemotherapeutic compound.
  • T cells e.g., engineered T cells
  • a "chemotherapeutic agent” or “chemotherapeutic compound” and their grammatical equivalents as used herein, can be a chemical compound useful in the treatment of cancer.
  • the chemotherapeutic cancer agents that can be used in combination with the disclosed T cell include, but are not limited to, mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, vindesine and NavelbineTM
  • chemotherapeutic cancer agents include topoisomerase I inhibitors, such as camptothecin compounds.
  • camptothecin compounds include CamptosarTM (irinotecan HCL), HycamtinTM (topotecan HCL) and other compounds derived from camptothecin and its analogues.
  • podophyllotoxin derivatives such as etoposide, teniposide and mitopodozide.
  • the present disclosure further encompasses other chemotherapeutic cancer agents known as alkylating agents, which alkylate the genetic material in tumor cells.
  • alkylating agents include without limitation cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, belustine, uracil mustard, chlomaphazin, and dacarbazine.
  • alkylating agents include without limitation cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, belustine, uracil mustard, chlomaphazin, and dacarbazine.
  • antimetabolites as chemotherapeutic agents. Examples of these types of agents include cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, azathioprime, and procarbazine.
  • chemotherapeutic cancer agents that may be used in the methods and compositions disclosed herein include antibiotics. Examples include without limitation doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mytomycin C, and daunomycin. There are numerous liposomal formulations commercially available for these compounds. The present disclosure further encompasses other chemotherapeutic cancer agents including without limitation anti-tumor antibodies, dacarbazine, azacytidine, amsacrine, melphalan, ifosfamide and mitoxantrone.
  • the disclosed T cell herein can be administered in combination with other anti-tumor agents,
  • Cytotoxic/antineoplastic agents can be defined as agents who attack and kill cancer cells.
  • Some cytotoxic/anti-neoplastic agents can be alkylating agents, which alkylate the genetic material in tumor cells, e.g., cis-platin,
  • cytotoxic/antineoplastic agents can be antimetabolites for tumor cells, e.g., cytosine arabinoside, fluorouracil, methotrexate, mercaptopuirine, azathioprime, and procarbazine.
  • cytotoxic/anti-neoplastic agents can be antibiotics, e.g., doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mytomycin C, and daunomycin.
  • antibiotics e.g., doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mytomycin C, and daunomycin.
  • cytotoxic/anti-neoplastic agents can be mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine and etoposide.
  • Miscellaneous cytotoxic/anti-neoplastic agents include taxol and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacytidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.
  • Anti-angiogenic agents can also be used. Suitable anti-angiogenic agents for use in the disclosed methods and compositions include anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides. Other inhibitors of angiogenesis include angiostatin, endostatin, interferons, interleukin 1 (including a and ⁇ ) interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinase-1 and -2. (TIMP-1 and -2). Small molecules, including topoisomerases such as razoxane, a topoisomerase II inhibitor with anti-angiogenic activity, can also be used.
  • anti-cancer agents that can be used in combination with the disclosed T cell include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;
  • carmustine carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin;
  • daunorubicin hydrochloride decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;
  • diaziquone diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin;
  • enloplatin enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; trasrabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-nl; interferon alfa-n3; interferon beta-I
  • masoprocol maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane;
  • mitoxantrone hydrochloride mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin;
  • riboprine riboprine; rogletimide; safingol; safingol hydrochloride; semustine; pumprazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin;
  • anti-cancer drugs include, but are not limited to: 20-epi-
  • adozelesin aldesleukin
  • ALL-TK antagonists altretamine
  • ambamustine amidox
  • amifostine
  • antiandrogen prostatic carcinoma
  • antiestrogen antineoplaston
  • antisense oligonucleotides aphidicolin glycinate
  • apoptosis gene modulators apoptosis regulators
  • apurinic acid ara-CDP-DL-
  • PTBA arginine deaminase
  • asulacrine asulacrine
  • atamestane atrimustine
  • axinastatin 1 axinastatin 2;
  • axinastatin 3 azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;
  • BCR/ABL antagonists benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;
  • calcipotriol calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide- amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam;
  • cypemycin cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine;
  • dehydrodidemnin B deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
  • diaziquone didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-;
  • dioxamycin diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; trasrabine; fenretinide; filgrastim;
  • finasteride flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin;
  • ilmofosine ilomastat
  • imidazoacridones imiquimod
  • immunostimulant peptides insulin-like growth factor-1 receptor inhibitor
  • interferon agonists interferons
  • interleukins interferons
  • iobenguane interleukins
  • iododoxorubicin ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon;
  • leuprolide+estrogen+progesterone leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin;
  • nartograstim nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives;
  • perfosfamide perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; piloca ⁇ ine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A- based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins;
  • pyrazoloacridine pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;
  • rohitukine romurtide; roquinimex; rubiginone B l; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1;
  • squalamine stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene;
  • thrombopoietin mimetic thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin;
  • toremifene totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
  • the anti -cancer drug is 5-fluorouracil, taxol, or leucovorin.
  • the unit dosage of the composition or formulation administered can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg.
  • the total amount of the composition or formulation administered can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 g.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a T cell can be administered either alone or together with a pharmaceutically acceptable carrier or excipient, by any routes, and such administration can be carried out in both single and multiple dosages.
  • the pharmaceutical composition can be combined with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hand candies, powders, sprays, aqueous suspensions, injectable solutions, elixirs, syrups, and the like.
  • Such carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, etc.
  • such oral pharmaceutical formulations can be suitably sweetened and/or flavored by means of various agents of the type commonly employed for such purposes.
  • cells can be administered to a patient in conjunction with (e.g. , before, simultaneously, or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, or Cytarabine (also known as ARA-C).
  • agents such as antiviral therapy, cidofovir and interleukin-2, or Cytarabine (also known as ARA-C).
  • the engineered cells can be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation.
  • immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies
  • immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies
  • cytoxin fludaribine
  • cyclosporin FK506, rapamycin
  • mycoplienolic acid steroids
  • steroids FR901228
  • cytokines irradiation
  • the engineered cell composition can also be administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
  • chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
  • the engineered cell compositions of the present invention can be administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.
  • subjects can undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell
  • the engineered cells e.g., expanded engineered cells
  • expanded engineered cells can be administered before or following surgery.
  • the engineered cells obtained by any one of the methods described herein can be used in a particular aspect of the invention for treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD). Therefore, a method of treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD) comprising treating a patient by administering to a patient an effective amount of engineered cells comprising inactivated TCR alpha and/or TCR beta genes is contemplated.
  • Cells can be extracted from a human as described herein. Cells can be genetically altered ex vivo and used accordingly. These cells can be used for cell-based therapies. These cells can be used to treat disease in a recipient (e.g., a human). For example, these cells can be used to treat cancer.
  • Described herein is a method of treating a disease (e.g., cancer) in a recipient comprising
  • Cells prepared by intracellular genomic transplant can be used to treat cancer.
  • Described herein is a method of treating a disease (e.g., cancer) in a recipient comprising
  • 5xl0 10 cells will be administered to a patient.
  • 5xl0 n cells will be administered to a patient.
  • about 5xl0 10 cells are administered to a subject. In some embodiments, about
  • 5xl0 10 cells represents the median amount of cells administered to a subject. In some embodiments, about 5xl0 10 cells are necessary to effect a therapeutic response in a subject. In some embodiments, at least about at least about lxlO 7 cells, at least about 2xl0 7 cells, at least about 3xl0 7 cells, at least about
  • 4xl0 7 cells at least about 5xl0 7 cells, at least about 6xl0 7 cells, at least about 6xl0 7 cells, at least about 8xl0 7 cells, at least about 9xl0 7 cells, at least about lxlO 8 cells, at least about 2xl0 8 cells, at least about 3xl0 8 cells, at least about 4xl0 8 cells, at least about 5xl0 8 cells, at least about 6xl0 8 cells, at least about 6xl0 8 cells, at least about 8xl0 8 cells, at least about 9xl0 8 cells, at least about lxlO 9 cells, at least about 2xl0 9 cells, at least about 3xl0 9 cells, at least about 4xl0 9 cells, at least about
  • 2xlO n cells at least about 3xl0 n cells, at least about 4xlO n cells, at least about 5xl0 n cells, at least about 6xlO n cells, at least about 6xlO n cells, at least about 8xl0 n cells, at least about 9xlO n cells, or at least about lxlO 12 cells.
  • about 5xl0 10 cells may be administered to a subject.
  • the cells may be expanded to about 5xl0 10 cells and administered to a subject.
  • cells are expanded to sufficient numbers for therapy.
  • 5 xlO 7 cells can undergo rapid expansion to generate sufficient numbers for therpauetic use.
  • sufficient numbers for therapeutic use can be 5xl0 10 .
  • Any number of cells can be infused for therapeutic use.
  • a patient may be infused with a numer of cells between lxl 0 6 to 5x10 12 inclusive.
  • a patient may be infused with as many cells that can be generated for them.
  • cells that are infused into a patient are not all engineered. For example, at least 90% of cells that are infused into a patient can be engineered. In other instances, at least 40% of cells that are infused into a patient can be engineered.
  • a method of the present disclosure comprises calculating and/or administering to a subject an amount of engineered cells necessary to effect a therapeutic response in the subject.
  • calculating the amount of engineered cells necessary to effect a therapeutic response comprises the viability of the cells and/or the efficiency with which a transgene has been integrated into the genome of a cell.
  • the cells administered to the subject may be viable cells.
  • the cells administered to a subject may be cells that have had one or more transgenes successfully integrated into the genome of the cell.
  • the method disclosed herein can be used for treating or preventing disease including, but not limited to, cancer, cardiovascular diseases, lung diseases, liver diseases, skin diseases, or neurological diseases.
  • Transplanting can be by any type of transplanting.
  • Sites can include, but not limited to, liver
  • transplanting can be subcapsular transplanting.
  • Transplanting can also be intramuscular transplanting.
  • Transplanting can be intraportal transplanting.
  • Transplanting can be of one or more cells from a human.
  • the one or more cells can be from an organ, which can be a brain, heart, lungs, eye, stomach, pancreas, kidneys, liver, intestines, uterus, bladder, skin, hair, nails, ears, glands, nose, mouth, lips, spleen, gums, teeth, tongue, salivary glands, tonsils, pharynx, esophagus, large intestine, small intestine, rectum, anus, thyroid gland, thymus gland, bones, cartilage, tendons, ligaments, suprarenal capsule, skeletal muscles, smooth muscles, blood vessels, blood, spinal cord, trachea, ureters, urethra, hypothalamus, pituitary, pylorus, adrenal glands, ovaries, oviducts, uterus, vagina, mammary glands, testes, seminal vesicles, penis, lymph, lymph no
  • the one or more cells can also be from a brain, heart, liver, skin, intestine, lung, kidney, eye, small bowel, or pancreas.
  • the one or more cells can be from a pancreas, kidney, eye, liver, small bowel, lung, or heart.
  • the one or more cells can be from a pancreas.
  • the one or more cells can be pancreatic islet cells, for example, pancreatic ⁇ cells.
  • the one or more cells can be any blood cells, such as peripheral blood mononuclear cell (PBMC), lymphocytes, monocytes or macrophages.
  • PBMC peripheral blood mononuclear cell
  • the one or more cells can be any immune cells such as lymphocytes, B cells, or T cells.
  • the method disclosed herein can also comprise transplanting one or more cells, where the one or more cells can be can be any types of cells.
  • the one or more cells can be epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, pancreatic islet cells, blood cells, blood precursor cells, bone cells, bone precursor cells, neuronal stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular endothelial cells, fibroblasts, liver stellate cells, aortic smooth muscle cells, cardiac myocytes, neurons, Kupffer cells, smooth muscle cells,
  • the one or more cells can be pancreatic islet cells and/or cell clusters or the like, including, but not limited to pancreatic a cells, pancreatic ⁇ cells, pancreatic ⁇ cells, pancreatic F cells (e.g. , PP cells), or pancreatic ⁇ cells.
  • the one or more cells can be pancreatic a cells.
  • the one or more cells can be pancreatic ⁇ cells.
  • Donor can be at any stage of development including, but not limited to, fetal, neonatal, young and adult.
  • donor T cells can be isolated from adult human.
  • Donor human T cells can be under the age of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year(s).
  • T cells can be isolated from a human under the age of 6 years.
  • T cells can also be isolated from a human under the age of 3 years.
  • a donor can be older than 10 years,
  • the method disclosed herein can comprise transplanting.
  • Transplanting can be auto transplanting, allotransplanting, xenotransplanting, or any other transplanting.
  • transplanting can be xenotransplanting.
  • Transplanting can also be allotransplanting.
  • Xenotransplantation and its grammatical equivalents as used herein can encompass any procedure that involves transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, where the recipient and donor are different species. Transplantation of the cells, organs, and/or tissues described herein can be used for xenotransplantation in into humans. Xenotransplantation includes but is not limited to vascularized xenotransplant, partially vascularized xenotransplant, unvascularized xenotransplant, xenodressings, xenobandages, and xenostructures.
  • Allotransplantation and its grammatical equivalents (e.g. , allogenic transplantation) as used herein can encompass any procedure that involves transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, where the recipient and donor are the same species but different individuals. Transplantation of the cells, organs, and/or tissues described herein can be used for allotransplantation into humans. Allotransplantation includes but is not limited to vascularized allotransplant, partially vascularized allotransplant, unvascularized allotransplant, allodressings, allobandages, and allostructures.
  • herein can encompass any procedure that involves transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, where the recipient and donor is the same individual.
  • Autotransplantation into humans.
  • Autotransplantation includes but is not limited to vascularized autotransplantation, partially vascularized autotransplantation, unvascularized autotransplantation, autodressings, autobandages, and autostructures.
  • transplant rejection can be improved as compared to when one or more wild-type cells is transplanted into a recipient.
  • transplant rejection can be hyperacute rejection.
  • Transplant rejection can also be acute rejection.
  • Other types of rejection can include chronic rejection.
  • Transplant rejection can also be cell-mediated rejection or T cell-mediated rejection.
  • Transplant rejection can also be natural killer cell-mediated rejection.
  • improving transplantation can mean lessening hyperacute rejection, which can encompass a decrease, lessening, or diminishing of an undesirable effect or symptom.
  • the transplanted cells can be functional in the recipient. Functionality can in some cases determine whether transplantation was successful.
  • the transplanted cells can be functional for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. This can indicate that transplantation was successful. This can also indicate that there is no rejection of the transplanted cells, tissues, and/or organs.
  • transplanted cells can be functional for at least 1 day.
  • Transplanted cells can also functional for at least 7 day.
  • Transplanted cells can be functional for at least 14 day.
  • Transplanted cells can be functional for at least 21 day.
  • Transplanted cells can be functional for at least 28 day.
  • Transplanted cells can be functional for at least 60 days.
  • Another indication of successful transplantation can be the days a recipient does not require
  • immunosuppressive therapy For example, after treatment (e.g. , transplantation) provided herein, a recipient can require no immunosuppressive therapy for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. This can indicate that transplantation was successful. This can also indicate that there is no rejection of the transplanted cells, tissues, and/or organs.
  • a recipient can require no immunosuppressive therapy for at least 1 day.
  • a recipient can also require no immunosuppressive therapy for at least 7 days.
  • a recipient can require no immunosuppressive therapy for at least 14 days.
  • a recipient can require no immunosuppressive therapy for at least 21 days.
  • a recipient can require no immunosuppressive therapy for at least 28 days.
  • a recipient can require no immunosuppressive therapy for at least 60 days.
  • a recipient can require no immunosuppressive therapy for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years.
  • Another indication of successful transplantation can be the days a recipient requires reduced
  • a recipient can require reduced immunosuppressive therapy for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. This can indicate that transplantation was successful. This can also indicate that there is no or minimal rejection of the transplanted cells, tissues, and/or organs.
  • a recipient can require no immunosuppressive therapy for at least 1 day.
  • a recipient can also require no immunosuppressive therapy for at least or at least about 7 days.
  • a recipient can require no immunosuppressive therapy for at least or at least about 14 days.
  • a recipient can require no immunosuppressive therapy for at least or at least about 21 days.
  • a recipient can require no immunosuppressive therapy for at least or at least about 28 days.
  • a recipient can require no immunosuppressive therapy for at least or at least about 60 days.
  • a recipient can require no immunosuppressive therapy for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years.
  • Another indication of successful transplantation can be the days a recipient requires reduced
  • a recipient can require reduced immunosuppressive therapy for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. This can indicate that transplantation was successful. This can also indicate that there is no or minimal rejection of the transplanted cells, tissues, and/or organs.
  • Reduced and its grammatical equivalents as used herein can refer to less immunosuppressive therapy compared to a required immunosuppressive therapy when one or more wild-type cells is transplanted into a recipient.
  • Immunosuppressive therapy can comprise any treatment that suppresses the immune system.
  • Immunosuppressive therapy can help to alleviate, minimize, or eliminate transplant rejection in a recipient.
  • immunosuppressive therapy can comprise immuno-suppressive drugs.
  • Immunosuppressive drugs that can be used before, during and/or after transplant, but are not limited to, MMF (mycophenolate mofetil (Cellcept)), ATG (anti-thymocyte globulin), anti-CD 154 (CD40L), anti-CD40 (2C10, ASKP1240, CCFZ533X2201), alemtuzumab (Campath), anti-CD20 (rituximab), anti-IL-6R antibody (tocilizumab, Actemra), anti-IL-6 antibody (sarilumab, olokizumab), CTLA4-Ig (Abatacept/Orencia), belatacept (LEA29Y), sirolimus (Rapimune), everolimus, tacrolimus (Prograf), daclizumab (Ze-napax), basiliximab (Simulect), infliximab (Remicade), cyclosporin,
  • MMF mycophenolate mofetil
  • one or more than one immunosuppressive agents/drugs can be used together or sequentially.
  • One or more than one immunosuppressive agents/drugs can be used for induction therapy or for maintenance therapy. The same or different drugs can be used during induction and maintenance stages.
  • daclizumab can be used for induction therapy and tacrolimus (Prograf) and sirolimus (Rapimune) can be used for maintenance therapy. Daclizumab (Zenapax) can also be used for induction therapy and low dose tacrolimus (Prograf) and low dose sirolimus (Rapimune) can be used for maintenance therapy.
  • Immunosuppression can also be achieved using non-drug regimens including, but not limited to, whole body irradiation, thymic irradiation, and full and/or partial splenectomy. These techniques can also be used in combination with one or more immuno-suppressive drugs.
  • Example 1 determine the transfection efficiency of various nucleic acid delivery platforms
  • PBMCs peripheral blood mononuclear cells
  • Leukopaks collected from normal peripheral blood were used herein. Blood cells were diluted 3 to 1 with chilled IX PBS. The diluted blood was added dropwise (e.g., very slowly) over 15 mLs of LYMPHOPREP (Stem Cell Technologies) in a 50 ml conical. Cells were spun at 400 x G for 25 minutes with no brake. The buffy coat was slowly removed and placed into a sterile conical. The cells were washed with chilled IX PBS and spun for 400 x G for 10 minutes. The supernatant was removed, cells resuspended in media, counted and viably frozen in freezing media (45 mLs heat inactivated FBS and 5 mLs DMSO).
  • PBMCs were thawed and plated for 1-2 hours in culturing media (RPMI-1640 (with no Phenol red), 20 % FBS (heat inactivated), and IX Gluta-MAX). Cells were collected and counted; the cell density was adjusted to 5 x 10 ⁇ 7 cells/mL and transferred to sterile 14 mL polystyrene round-bottom tube. Using the EasySep Human CD3 cell Isolation Kit (Stem Cell Technologies), 50 uL/mL of the Isolation Cocktail was added to the cells. The mixture was mixed by pipetting and incubated for 5 minutes at room temperature.
  • RPMI-1640 with no Phenol red
  • FBS heat inactivated
  • IX Gluta-MAX IX Gluta-MAX
  • RapidSpheres were vortexed for 30 seconds and added at 50 uL/mL to the sample; mixed by pipetting. Mixture was topped off to 5 mLs for samples less than 4 mLs or topped off to 10 mLs for samples more than 4 mLs.
  • the sterile polystyrene tube was added to the "Big Easy" magnet; incubated at room temperature for 3 minutes. The magnet and tube, in one continuous motion, were inverted, pouring off the enriched cell suspension into a new sterile tube.
  • Isolated CD3+ T cells were counted and plated out at a density of 2 x 10 ⁇ 6 cells/mL in a 24 well plate.
  • Dynabeads Human T-Activator CD3/CD28 beads (Gibco, Life Technologies) were added 3: 1 (beads: cells) to the cells after being washed with IX PBS with 0.2% BSA using a dynamagnet.
  • IL-2 (Peprotech) was added at a concentration of 300 IU/mL. Cells were incubated for 48 hours and then the beads were removed using a dynamagnet. Cells were cultured for an additional 6-12 hours before electroporation or nucelofection.
  • Unstimulated or stimulated T cells were nucleofected using the Amaxa Human T Cell Nucleofector Kit (Lonza, Switzerland), FIG. 82 A. and FIG. 82 B. Cells were counted and resuspended at of density of 1-8 x 10 ⁇ 6 cells in 100 uL of room temperature Amaxa buffer. 1-15 ug of mRNA or plasmids were added to the cell mixture. Cells were nucleofected using the U-014 program. After nucleofection, cells were plated in 2 mLs culturing media in a 6 well plate.
  • Unstimulated or stimulated T cells were electroporated using the Neon Transfection System (10 uL Kit, Invitrogen, Life Technologies). Cells were counted and resuspended at a density of 2 x 10 ⁇ 5 cells in 10 uL of T buffer. 1 ug of GFP plasmid or mRNA or 1 ug Cas9 and 1 ug of gRNA plasmid were added to the cell mixture. Cells were electroporated at 1400 V, 10 ms, 3 pulses. After transfection, cells were plated in a 200 uL culturing media in a 48 well plate.
  • Unstimulated T cells were plated at a density of 5 x 10 ⁇ 5 cells per mL in a 24 well plate.
  • T cells were transfected with 500 ng of mRNA using the TransIT-mRNA Transfection Kit (Mirus Bio), according to the manufacturer's protocol.
  • Plasmid DNA transfection the T cells were transfected with 500 ng of plasmid DNA using the TransIT-X2 Dynamic Delivery System (Mirus Bio), according to the manufacturer's protocol. Cells were incubated at 37°C for 48 hours before being analyzed by flow cytometry.
  • Unstimulated or stimulated T cells were plated at a density of 1-2 x 10 ⁇ 5 cells per well in a 48 well plate in 200 uL of culturing media.
  • Gold nanoparticle SmartFlared complexed to Cy5 or Cy3 (Millipore, Germany) were vortexed for 30 seconds prior to being added to the cells.
  • 1 uL of the gold nanoparticle SmartFlares was added to each well of cells. The plate was rocked for 1 minute incubated for 24 hours at 37°C before being analyzed for Cy5 or Cy3 expression by flow cytometry.
  • Electroporated and nucleofected T cells were analyzed by flow cytometry 24-48 hours post
  • the six cell and DNA/RNA combinations were: adding EGFP plasmid DNA to unstimulated PBMCs; adding EGFP plasmid DNA to unstimulated T cells; adding EGFP plasmid DNA to stimulated T cells; adding EGFP mRNA to unstimulated PBMCs; adding EGFP mRNA to unstimulated T cells; and adding EGFP mRNA to stimulated T cells.
  • the four exemplary transfection platforms were: AMAXA Nucleofection, NEON Eletrophoration, Lipid-based Transfection, and Gold Nanoparticle delivery. The transfection efficiency (% of transfected cells) results under various conditions were listed in Table 1 and adding mRNA to stimulated T cells using AMAXA platform provides the highest efficiency.

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