WO2018035141A1 - Il-10 receptor alpha homing endonuclease variants, compositions, and methods of use - Google Patents

Il-10 receptor alpha homing endonuclease variants, compositions, and methods of use Download PDF

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Publication number
WO2018035141A1
WO2018035141A1 PCT/US2017/046989 US2017046989W WO2018035141A1 WO 2018035141 A1 WO2018035141 A1 WO 2018035141A1 US 2017046989 W US2017046989 W US 2017046989W WO 2018035141 A1 WO2018035141 A1 WO 2018035141A1
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cell
polypeptide
gene
lora
cells
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PCT/US2017/046989
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French (fr)
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Kyle HAVENS
Jordan JARJOUR
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Bluebird Bio, Inc.
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Priority to EP17842000.6A priority Critical patent/EP3500696A4/en
Priority to US16/324,357 priority patent/US20190309274A1/en
Priority to CA3034094A priority patent/CA3034094A1/en
Publication of WO2018035141A1 publication Critical patent/WO2018035141A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • the present invention relates to improved genome editing compositions. More particularly, the invention relates to nuclease variants, compositions, and methods of using same for editing the human interleukin 10 receptor 1 alpha (IL-lORa) gene.
  • IL-lORa human interleukin 10 receptor 1 alpha
  • Interleukin- 10 is an immunomodulatory pleiotropic cytokine produced by B cells, T cells, and cells of the monocyte/macrophage lineage and exhibits diverse activities on various cell types in the immune system.
  • IL-10 signaling is mediated through an IL-IO/IL- lORa/ ⁇ receptor signaling complex and is associated with immunosuppression, whereas lack of IL-10 signaling is associated with autoimmune disease.
  • IL-10 signaling is associated with immunosuppressive tumor microenvironments and cancers having a poor prognosis, e.g., melanoma, multiple myeloma.
  • IL-10 signaling is associated with immunosuppressive tumor microenvironments and cancers having a poor prognosis, e.g., melanoma, multiple myeloma.
  • immunosuppression may limit the magnitude of T cell responses by inhibiting expression of MHC class II molecules, costimulatory molecules, and proinflammatory cytokines, e.g., including, but not limited to, tumor necrosis factor-alpha (TNFa), interleukin-6 (IL-6) and interleukin-1 beta (IL- ⁇ ), in antigen presenting cells (APCs).
  • TNFa tumor necrosis factor-alpha
  • IL-6 interleukin-6
  • IL- ⁇ interleukin-1 beta
  • IL-10 signaling also plays a role in immune cell function and homeostasis.
  • Immune cells that express IL- 1 OR have the ability to suppress immune responses, including
  • IL-lORa IL-lORa
  • gut macrophages are associated with susceptibility to autoimmune diseases.
  • disruption of IL-lORa in regulatory T cells (Tregs) deregulates Treg function and leads to severe autoimmune colitis.
  • the invention generally relates, in part, to compositions comprising homing endonuclease variants and megaTALs that cleave a target site in the human IL-lORa gene and methods of using the same.
  • the present invention contemplates, in part, a polypeptide comprising a homing endonuclease (HE) variant that cleaves a target site in the human interleukin 10 receptor 1 alpha (IL-lORa) gene.
  • HE homing endonuclease
  • the HE variant is an LAGLIDADG homing endonuclease (LHE) variant.
  • LHE LAGLIDADG homing endonuclease
  • the polypeptide comprises a biologically active fragment of the HE variant.
  • the biologically active fragment lacks the 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal amino acids compared to a corresponding wild type HE. In additional embodiments, the biologically active fragment lacks the 4 N-terminal amino acids compared to a corresponding wild type HE.
  • the biologically active fragment lacks the 8 N-terminal amino acids compared to a corresponding wild type HE.
  • the biologically active fragment lacks the 1, 2, 3, 4, or 5 C- terminal amino acids compared to a corresponding wild type HE.
  • the biologically active fragment lacks the C- terminal amino acid compared to a corresponding wild type HE.
  • the biologically active fragment lacks the 2 C-terminal amino acids compared to a corresponding wild type HE.
  • the HE variant is a variant of an LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, I-ApaMI, I-CapHI, I-CapIV, I-CkaMI, I-CpaMI, I- CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-Gpil, I- GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I- Ncrl, I-NcrMI, I-OheMI, I-Onul, I-OsoMI, I-OsoMII, I-OsoMni,
  • the HE variant is a variant of an LHE selected from the group consisting of: I-CpaMI, I-HjeMI, I-Onul, I-PanMI, and SmaMI.
  • the HE variant is an I-Onul LHE variant.
  • the HE variant comprises one or more amino acid substitutions in the DNA recognition interface at amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
  • the HE variant comprises at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions in the DNA recognition interface at amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
  • the HE variant comprises at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions in at least one position selected from the position group consisting of positions: 24, 26, 28, 30, 32, 34, 36, 37, 38, 40, 41, 42, 44, 46, 48, 59, 70, 72, 75, 78, 80, 82, 138, 143, 145, 159, 168, 180, 182, 184, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 207, 223, 225, 227, 228, 229, 232, 236, 238, and 240 of any one of SEQ ID NOs: 1-5, or a biologically active fragment thereof.
  • the HE variant comprises at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46V, Q46I, T48G, N59S, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180H, C180Y, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K,
  • the HE variant comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46V, T48G, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, S159P, F168L, C180H, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, K229A, F232K, D236K, and V238I, in reference to an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
  • the HE variant comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46I, T48G, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180H, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K, and V238I, in reference to an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
  • the HE variant comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46I, T48G, N59S, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180Y, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K, and V238I, in reference to an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically
  • the HE variant comprises an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 6-8, or a biologically active fragment thereof.
  • the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.
  • the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 7, or a biologically active fragment thereof.
  • the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 8, or a biologically active fragment thereof.
  • the polypeptide further comprises a DNA binding domain.
  • the DNA binding domain is selected from the group consisting of: a TALE DNA binding domain and a zinc finger DNA binding domain.
  • the TALE DNA binding domain comprises about 9.5 TALE repeat units to about 15.5 TALE repeat units.
  • the TALE DNA binding domain binds a polynucleotide sequence in the IL-lORa gene.
  • the TALE DNA binding domain binds the polynucleotide sequence set forth in SEQ ID NO: 11.
  • the polypeptide binds and cleaves the polynucleotide sequence set forth in SEQ ID NO: 13.
  • the zinc finger DNA binding domain comprises 2, 3, 4, 5, 6, 7, or 8 zinc finger motifs.
  • polypeptide further comprises a peptide linker and an end- processing enzyme or biologically active fragment thereof.
  • the polypeptide further comprises a viral self-cleaving 2A peptide and an end-processing enzyme or biologically active fragment thereof.
  • the end-processing enzyme or biologically active fragment thereof has 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease, 5 ' flap endonuclease, helicase or template-independent DNA polymerase activity.
  • the end-processing enzyme comprises Trex2 or a biologically active fragment thereof.
  • the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-11, or a biologically active fragment thereof.
  • polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 9, or a biologically active fragment thereof.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 10, or a biologically active fragment thereof.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 11, or a biologically active fragment thereof.
  • polypeptide cleaves the human IL-lORa gene at the polynucleotide sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 15.
  • the present invention contemplates, in part, a polynucleotide encoding a polypeptide contemplate herein.
  • the present invention contemplates, in part, an mRNA encoding a polypeptide contemplated herein.
  • the mRNA comprises the sequence set forth in SEQ ID NO: 19.
  • the present invention contemplates, in part, a cDNA encoding a polypeptide contemplated herein. In certain embodiments, the present invention contemplates, in part, a vector comprising a polynucleotide encoding a polypeptide contemplated herein.
  • the present invention contemplates, in part, a cell comprising a polypeptide contemplated herein.
  • the present invention contemplates, in part, a cell comprising a polynucleotide encoding a polypeptide contemplated herein.
  • the present invention contemplates, in part, a cell comprising a vector contemplated herein.
  • the present invention contemplates, in part, a cell comprising one or more genome modifications introduced by a polypeptide contemplated herein.
  • the cell is a hematopoietic cell.
  • the cell is a T cell.
  • the cell is a CD3+, CD4+, and/or CD8+ cell.
  • the cell is an immune effector cell.
  • the cell is a cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cells.
  • CTLs cytotoxic T lymphocytes
  • TILs tumor infiltrating lymphocytes
  • helper T cells a helper T cell.
  • the cell is a natural killer (NK) cell or natural killer T (KT) cell.
  • NK natural killer
  • KT natural killer T
  • the cell is a regulatory T cell (Treg).
  • the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
  • the cell comprises a polynucleotide encoding an engineered antigen receptor.
  • the engineered antigen receptor is selected from the group consisting of: an engineered T cell receptor, a chimeric antigen receptor, a DARIC, or a zetakine.
  • the cell comprises a polynucleotide encoding a bispecific T cell engager (BiTE) molecule; a hormone; a cytokine (e.g., IL-2, insulin, IFN- ⁇ , IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-a), a chemokine (e.g., ⁇ - ⁇ , ⁇ - ⁇ , MCP-1, MCP-3, and RANTES), a cytotoxin (e.g., Perforin, Granzyme A, and Granzyme B), or a cytokine receptor (e.g., an JL-2 receptor, an IL-7 receptor, an IL-12 receptor, or an IL-15 receptor, and an IL-21 receptor).
  • a cytokine e.g., IL-2, insulin, IFN- ⁇ , IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-a
  • a chemokine e.
  • the cell comprises a polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
  • the polynucleotide is integrated into the IL-lORa gene.
  • the polynucleotide is a donor repair template integrated into the IL- 1 ORa gene at a DNA double stranded break site introduced by the polypeptide
  • the polynucleotide is a donor repair template designed to correct one or more loss-of-function mutations in the endogenous IL-lORa gene, and wherein the donor repair template is integrated into the IL-lORa gene at a DNA double stranded break site introduced by the polypeptide contemplated herein.
  • IL-lORa expression is maintained, restored or increased and the polynucleotide encodes FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
  • the present invention contemplates, in part, a plurality of cells comprising one or more cells contemplated herein.
  • the present invention contemplates, in part, a composition comprising one or more cells contemplated herein.
  • the present invention contemplates, in part, a composition comprising one or more cells contemplated herein and a physiologically acceptable carrier.
  • the present invention contemplates, in part, a method of editing a human IL-lORa gene in a cell comprising: introducing a polynucleotide encoding a polypeptide contemplate herein into the cell, wherein expression of the polypeptide creates a double strand break at a target site in a human IL-lORa gene.
  • the present invention contemplates, in part, a method of editing a human IL-lORa gene in cell comprising: introducing a polynucleotide encoding a polypeptide contemplated herein into the cell, wherein expression of the polypeptide creates a double strand break at a target site in a human IL-lORa gene, wherein the break is repaired by non-homologous end joining (HEJ).
  • HEJ non-homologous end joining
  • the present invention contemplates, in part, a method of editing a human IL-lORa gene in a cell comprising: introducing a polynucleotide encoding a polypeptide contemplated herein and a donor repair template into the cell, wherein expression of the polypeptide creates a double strand break at a target site in a human IL-lORa gene and the donor repair template is incorporated into the human IL-lORa gene by homology directed repair (HDR) at the site of the double-strand break (DSB).
  • HDR homology directed repair
  • IL-lORa expression is maintained, restored or increased and the polynucleotide encodes FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
  • the cell is a hematopoietic cell.
  • the cell is a T cell.
  • the cell is a CD3+, CD4+, and/or CD8+ cell.
  • the cell is an immune effector cell.
  • the cell is a cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cells.
  • CTLs cytotoxic T lymphocytes
  • TILs tumor infiltrating lymphocytes
  • helper T cells a helper T cell.
  • the cell is a natural killer (NK) cell or natural killer T (NKT) cell.
  • NK natural killer
  • NKT natural killer T
  • the cell is a regulatory T cell (Treg).
  • the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
  • the polynucleotide encoding the polypeptide is an mRNA.
  • a polynucleotide encoding a 5 -3 ' exonuclease is introduced into the cell.
  • a polynucleotide encoding Trex2 or a biologically active fragment thereof is introduced into the cell.
  • the donor repair template encodes a FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
  • the donor repair template encodes a wild type copy of the IL- 1 ORa gene or portion thereof.
  • the donor repair template encodes an IL-lORa gene or portion thereof comprising one or more mutations compared to the wild type IL-lORa gene.
  • the donor repair template encodes an engineered antigen receptor.
  • the engineered antigen receptor is selected from the group consisting of: an engineered T cell receptor, a chimeric antigen receptor, a DARIC, or a zetakine.
  • the donor repair template encodes a bispecific T cell engager (BiTE) molecule; a hormone; a cytokine (e.g., IL-2, insulin, IFN- ⁇ , IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-a), a chemokine (e.g., ⁇ - ⁇ , ⁇ - ⁇ , MCP-1, MCP-3, and RANTES), a cytotoxin (e.g., Perforin, Granzyme A, and Granzyme B), or a cytokine receptor (e.g., an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, or an IL-15 receptor, and an IL-21 receptor).
  • a cytokine e.g., IL-2, insulin, IFN- ⁇ , IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-a
  • a chemokine e.g., ⁇ - ⁇ ,
  • the donor repair template comprises a 5 ' homology arm homologous to a human IL-lORa gene sequence 5 ' of the DSB and a 3 ' homology arm homologous to a human IL- 1 ORa gene sequence 3 ' of the D SB .
  • the lengths of the 5 ' and 3 ' homology arms are independently selected from about 100 bp to about 2500 bp.
  • the lengths of the 5 ' and 3 ' homology arms are independently selected from about 600 bp to about 1500 bp.
  • the 5 ' homology arm is about 1500 bp and the 3 ' homology arm is about 1000 bp.
  • the 5 ' homology arm is about 600 bp and the 3 ' homology arm is about 600 bp.
  • a viral vector is used to introduce the donor repair template into the cell.
  • the viral vector is a recombinant adeno-associated viral vector (rAAV) or a retrovirus.
  • the rAAV has one or more ITRs from AAV2.
  • the rAAV has a serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.
  • the rAAV has an AAV2 or AAV6 serotype.
  • the retrovirus is a lentivirus.
  • the lentivirus is an integrase deficient lentivirus (IDLV).
  • IDLV integrase deficient lentivirus
  • the present invention contemplates, in part, a method of treating, preventing, or ameliorating at least one symptom of a cancer, GVHD, transplant rejection, infectious disease, autoimmune disease, inflammatory disease, and
  • the present invention contemplates, in part, a method of treating a solid cancer comprising administering to the subj ect an effective amount of a composition contemplated herein.
  • the solid cancer comprises liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.
  • the present invention contemplates, in part, a method of treating a hemotological malignancy comprising administering to the subject an effective amount of a composition contemplated herein.
  • the hematological malignancy is a leukemia, lymphoma, or multiple myeloma.
  • the present invention contemplates, in part, a method of treating an autoimmune disease comprising administering to the subject an effective amount of a composition contemplated herein.
  • the autoimmune disease is associated with a loss-of-function mutation in the IL- 1 ORa gene.
  • the loss-of-function mutation is a missense mutation, nonsense mutation, or splice site mutation.
  • the autoimmune disease is arthritis.
  • the autoimmune disease is inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the IBD is selected from the group consisting of ulcerative colitis, early onset ulcerative colitis, very early onset ulcerative colitis, pancolitis, Crohn's disease, and neonatal-onset Crohn's disease.
  • the autoimmune disease is associated with a loss-of-function mutation in the IL-lORa gene selected from the group consisting of: W45G; Y64C; W69R; T84I; Y91C; V100G; R101W; R117H; S138G; G141R; I169T; c.537G> A, p.T179T;
  • the present invention contemplates, in part, a method of treating graft- versus-host disease (GVHD) comprising administering to the subject an effective amount of a composition contemplated herein.
  • GVHD graft- versus-host disease
  • the GVHD is associated with a solid organ transplant in the subject.
  • the solid organ transplant is selected from the group consisting of: a heart transplant, a lung transplant, a kidney transplant, a pancreas transplant, and a liver transplant.
  • the present invention contemplates, in part, a method of preventing graft-versus-host disease (GVHD) while maintaining a graft-versus-leukemia response comprising administering to the subject an effective amount of a composition contemplated herein.
  • GVHD graft-versus-host disease
  • the GVHD is associated with a bone marrow transplant in the subject.
  • Figure 1 shows the human IL-lORa gene and the location of the homing endonuclease target site within exon 2 (SEQ ID NOs: 60 and 61).
  • Figure 2 shows reprogramming of the I-Onul N-terminal domain (NTD) and C- terminal domain (CTD) against chimeric "half-sites" through three rounds of sorting, followed by fusion of the reprogrammed domains to isolate a fully reprogrammed I-Onul homing endonuclease that cleaves the target site.
  • NTD I-Onul N-terminal domain
  • CCD C- terminal domain
  • Figure 3 shows the initial screening of I-Onul derived homing endonuclease variants for activity against an IL-lORa target site in a chromosomal reporter assay.
  • Figure 3 also shows the refinement of the initially derived I-Onul derived homing endonuclease IL-10Ra.G7 to achieve a more active variants, IL-10Ra.G7.A3 and IL-10Ra.G7.A3.G7.
  • Figure 4 shows that the IL-10Ra.G7.A3.G7 homing endonuclease has sub-nanomolar affinity properties as measured using a yeast surface display based substrate titration assay.
  • Figure 5 shows an alignment of IL-10Ra.G7 (SEQ ID NO: 63), IL-10Ra.G7.A3 (SEQ ID NO: 64) and IL-10Ra.G7.A3.G7 (SEQ ID NO: 65) homing endonucleases compared to the wild type I-Onul (SEQ ID NO: 62) homing endonucleases, highlighting non-identical positions.
  • Figure 6 A shows a schematic of an IL-lORa megaTAL (SEQ ID NO: 67) that targets the IL-lORa gene (SEQ ID NO: 66).
  • Figure 6B shows a TIDE analysis of the genome editing of IL-lORa megaTAL co- delivered with Trex2 of the IL-lORa target sequence in primary human T cells.
  • Figure 7A shows a schematic of an AAV donor repair template that targets the IL- lORa gene.
  • Figure 7B shows that T cells treated with IL-lORa megaTAL and AAV donor repair template undergo HDR.
  • SEQ ID NO: 1 is an amino acid sequence of a wild type I-Onul LAGLIDADG homing endonuclease (LHE).
  • SEQ ID NO: 2 is an amino acid sequence of a wild type I-Onul LHE.
  • SEQ ID NO: 3 is an amino acid sequence of a biologically active fragment of a wild- type I-Onul LHE.
  • SEQ ID NO: 4 is an amino acid sequence of a biologically active fragment of a wild- type I-Onul LHE.
  • SEQ ID NO: 5 is an amino acid sequence of a biologically active fragment of a wild- type I-Onul LHE.
  • SEQ ID NO: 6 is an amino acid sequence of an I-Onul LHE variant reprogrammed to bind and cleave a target site in the human IL-lORa gene.
  • SEQ ID NO: 7 is an amino acid sequence of an I-Onul LHE variant reprogrammed to bind and cleave a target site in the human IL-lORa gene.
  • SEQ ID NO: 8 is an amino acid sequence of an I-Onul LHE variant reprogrammed to bind and cleave a target site in the human IL- 1 ORa gene.
  • SEQ ID NO: 9 is an amino acid sequence of a megaTAL that binds and cleaves a target site in a human IL-lORa gene.
  • SEQ ID NO: 10 is an amino acid sequence of a megaTAL that binds and cleaves a target site in a human IL-lORa gene.
  • SEQ ID NO: 11 is an amino acid sequence of a megaTAL that binds and cleaves a target site in a human IL-lORa gene.
  • SEQ ID NO: 12 is an amino acid sequence of a megaTAL-Trex2 fusion protein that binds and cleaves a target site in a human IL-lORa gene.
  • SEQ ID NO: 13 is an I-Onul LHE variant target site in a human IL-lORa gene.
  • SEQ ID NO: 14 is a TALE DNA binding domain target site in a human IL- 1 ORa gene.
  • SEQ ID NO: 15 is a megaTAL target site in a human IL-lORa gene.
  • SEQ ID NO: 16 is an I-Onul LHE variant N-terminal domain target site.
  • SEQ ID NO: 17 is an I-Onul LHE variant C-terminal domain target site.
  • SEQ ID NO: 18 is a polynucleotide sequence of an I-Onul LHE variant surface display plasmid.
  • SEQ ID NO: 19 is an mRNA sequence encoding a megaTAL that cleaves a human IL- lORa gene.
  • SEQ ID NO: 20 is an mRNA sequence encoding murine Trex2.
  • SEQ ID NO: 21 is an amino acid sequence encoding murine Trex2.
  • SEQ ID NO: 22 is an AAV-based ILlORa pMND-GFP donor repair template.
  • SEQ ID Nos: 23-33 forth the amino acid sequences of various linkers.
  • SEQ ID NOs: 34-58 set forth the amino acid sequences of protease cleavage sites and self-cleaving polypeptide cleavage sites.
  • X refers to any amino acid or the absence of an amino acid.
  • the invention generally relates to, in part, improved genome editing compositions and methods of use thereof.
  • genome editing compositions contemplated in various embodiments can be used to prevent or treat a cancer, graft- versus-host-disease (GVHD), transplant rejection, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or condition associated therewith, or ameliorates at least one symptom thereof.
  • GVHD graft- versus-host-disease
  • transplant rejection infectious disease
  • autoimmune disease inflammatory disease
  • immunodeficiency or condition associated therewith
  • Genome editing methods contemplated in particular embodiments are realized, in part, through modification of the IL-10 receptor, IL-lORa.
  • the immunosuppressive effects of IL- 10/IL-lORa signaling in the tumor microenvironment limit the effectiveness of adoptive immunotherapies.
  • defects in IL-10 signaling mediated through IL-lORa are associated with a compromised ability to respond to inflammatory disease and autoimmune disorders.
  • Genome editing compositions and methods contemplated in various embodiments comprise nuclease variants, designed to bind and cleave a target site in the human interleukin 10 receptor 1 alpha (IL-lORa) gene.
  • the nuclease variants contemplated in particular embodiments can be used to introduce a double-strand break in a target polynucleotide sequence, which may be repaired by non-homologous end joining (HEJ) in the absence of a polynucleotide template, e.g., a donor repair template, or by homology directed repair (HDR), i.e., homologous recombination, in the presence of a donor repair template.
  • HEJ non-homologous end joining
  • HDR homology directed repair
  • Nuclease variants contemplated in certain embodiments can also be designed as nickases, which generate single- stranded DNA breaks that can be repaired using the cell ' s base-excision-repair (BER) machinery or homologous recombination in the presence of a donor repair template.
  • NHEJ is an error-prone process that frequently results in the formation of small insertions and deletions that disrupt gene function.
  • Homologous recombination requires homologous DNA as a template for repair and can be leveraged to create a limitless variety of modifications specified by the introduction of donor DNA containing the desired sequence at the target site, flanked on either side by sequences bearing homology to regions flanking the target site.
  • the genome editing compositions contemplated herein comprise a homing endonuclease variant or megaTAL that targets the human IL-lORa gene.
  • the genome editing compositions contemplated herein comprise a homing endonuclease variant or megaTAL and an end-processing enzyme, e.g., Trex2.
  • genome edited cells are contemplated.
  • the genome edited cells comprise an edited IL-lORa gene, wherein the editing strategy is designed to decrease or eliminate IL-lORa expression or wherein the editing strategy is designed to increase or restore expression of IL-lORa by correcting one or more mutations in the IL-lORa gene.
  • the genome editing strategy comprises introducing a
  • polynucleotide in the IL-lORa gene without disrupting IL-lORa expression encodes a polypeptide that enhances Treg function.
  • a DNA break is generated in a target site of the IL-lORa gene in a T cell or immune effector cell, and NHEJ of the ends of the cleaved genomic sequence may result in a cell with little or no IL-lORa expression, and preferably a T cell that lacks or substantially lacks functional IL-lORa expression, e.g., lacks the ability to increase T cell exhaustion and to inhibit expression of MHC class ⁇ molecules, costimulatory molecules, and proinflammatory cytokines.
  • T cells that lack IL-lORa expression are more resistant to immunosuppression and T cell exhaustion, and thus, are more therapeutically efficacious.
  • a donor template for repair of the cleaved IL-lORa genomic sequence is provided.
  • the IL-lORa gene is repaired with the sequence of the template by homologous recombination at the DNA break-site.
  • the repair template comprises a polynucleotide sequence that is different from a targeted genomic sequence.
  • the repair template comprises a polynucleotide encoding an IL-lORa sequence that restores IL-lORa function.
  • a donor template encoding a modified IL-lORa polypeptide may be used to repair the cleaved IL-lORa genomic sequence.
  • the IL-lORa gene is repaired with the sequence of the template by homologous recombination at the DNA break- site.
  • the repair template comprises a polynucleotide encoding an IL-lORa sequence that modifies IL-lORa function, by increasing or decreasing receptor signaling, e.g., by modifying the affinity of IL-lORa for its cognate ligand IL-10.
  • a DNA break is generated in a target site of the IL- 1 ORa gene in a T cell or regulatory T cell (Treg), and donor template encoding a polypeptide that enhances Treg function is introduced into the IL-lORa gene at the double-stranded DNA break, without disrupting expression of IL-lORa.
  • Treg regulatory T cell
  • T cells or Tregs that comprise an edited IL-lORa gene comprising a polynucleotide encoding a polypeptide that enhances Treg function expressed from the IL- 10Ra promoter or an exogenous promoter inserted into the IL-lORa gene are more stable Tregs that are more therapeutically efficacious in maintaining graft-versus-leukemia (GVL) activity, e.g., post bone marrow or solid organ transplants, preventing transplant rejections, e.g., from bone marrow or solid organ transplants, treating graft-versus-host-disease (GVHD), e.g., resulting from bone marrow or solid organ transplants, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or a condition associated therewith.
  • VTL graft-versus-leukemia
  • GVHD graft-versus-host-disease
  • the genome editing compositions and methods are provided.
  • the genome editing compositions and methods are provided.
  • contemplated herein are used to edit the human IL-lORa gene.
  • compositions contemplated herein represent a quantum improvement compared to existing adoptive cell therapies.
  • an element means one element or one or more elements.
  • the term "about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the term "about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%>, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • a range e.g., 1 to 5, about 1 to 5, or about 1 to about 5, refers to each numerical value encompassed by the range.
  • the range "1 to 5" is equivalent to the expression 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.
  • the term “substantially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is 80%>, 85%>, 90%>, 91%>, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • “substantially the same” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that produces an effect, e.g., a physiological effect, that is approximately the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • ex vivo refers generally to activities that take place outside an organism, such as experimentation or measurements done in or on living tissue in an artificial environment outside the organism, preferably with minimum alteration of the natural conditions.
  • "ex vivo" procedures involve living cells or tissues taken from an organism and cultured or modulated in a laboratory apparatus, usually under sterile conditions, and typically for a few hours or up to about 24 hours, but including up to 48 or 72 hours, depending on the circumstances.
  • tissues or cells can be collected and frozen, and later thawed for ex vivo treatment. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissue are typically considered to be "in vitro " though in certain embodiments, this term can be used interchangeably with ex vivo.
  • in vivo refers generally to activities that take place inside an organism.
  • cellular genomes are engineered, edited, or modified in vivo.
  • “enhance” or “promote” or “increase” or “expand” or “potentiate” refers generally to the ability of a nuclease variant, genome editing composition, or genome edited cell contemplated herein to produce, elicit, or cause a greater response ⁇ i.e., physiological response) compared to the response caused by either vehicle or control.
  • a measurable response may include an increase in catalytic activity, binding affinity, persistence, in cytolytic activity, and/or an increase in proinflammatory cytokines, among others apparent from the
  • An "increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or control.
  • dampen refers generally to the ability of a nuclease variant, genome editing composition, or genome edited cell contemplated herein to produce, elicit, or cause a lesser response (i.e., physiological response) compared to the response caused by either vehicle or control.
  • a measurable response may include a decrease in off-target binding affinity, off- target cleavage specificity, anti-inflammatory cytokine production and/or secretion, and the like.
  • a “decrease” or “reduced” amount is typically a “statistically significant” amount, and may include an decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle, or control.
  • nuclease variant, genome editing composition, or genome edited cell contemplated herein refers generally to the ability of a nuclease variant, genome editing composition, or genome edited cell contemplated herein to produce, elicit, or cause a substantially similar or comparable physiological response (i.e., downstream effects) in as compared to the response caused by either vehicle or control.
  • a comparable response is one that is not significantly different or measurable different from the reference response.
  • binding affinity or “specifically binds” or “specifically bound” or “specific binding” or “specifically targets” as used herein, describe binding of one molecule to another, e.g., DNA binding domain of a polypeptide binding to DNA, at greater binding affinity than background binding.
  • a binding domain “specifically binds” to a target site if it binds to or associates with a target site with an affinity or K a (i.e., an equilibrium association constant of a particular binding interaction with units of l/M) of, for example, greater than or equal to about 10 5 M "1 .
  • a binding domain binds to a target site with a K a greater than or equal to about 10 6 M “1 , 10 7 M “1 , 10 8 M “1 , 10 9 M “1 , 10 10 M “1 , 10 11 M “1 , 10 12 M “ 1 , or 10 13 M “1 .
  • "High affinity” binding domains refers to those binding domains with a K a of at least 10 7 M “1 , at least 10 8 M “1 , at least 10 9 M “1 , at least 10 10 M “1 , at least 10 11 M “1 , at least 10 12 M “1 , at least 10 13 M “1 , or greater.
  • affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10 "5 M to 10 "13 M, or less).
  • Kd equilibrium dissociation constant
  • Affinities of nuclease variants comprising one or more DNA binding domains for DNA target sites contemplated in particular embodiments can be readily determined using conventional techniques, e.g., yeast cell surface display, or by binding association, or displacement assays using labeled ligands.
  • the affinity of specific binding is about 2 times greater than background binding, about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.
  • selectively binds or “selectively bound” or “selectively binding” or “selectively targets” and describe preferential binding of one molecule to a target molecule (on- target binding) in the presence of a plurality of off-target molecules.
  • an HE or megaTAL selectively binds an on-target DNA binding site about 5, 10, 15, 20, 25, 50, 100, or 1000 times more frequently than the HE or megaTAL binds an off-target DNA target binding site.
  • On-target refers to a target site sequence.
  • Off-target refers to a sequence similar to but not identical to a target site sequence.
  • a “target site” or “target sequence” is a chromosomal or extrachromosomal nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind and/or cleave, provided sufficient conditions for binding and/or cleavage exist.
  • a polynucleotide sequence or SEQ ID NO. that references only one strand of a target site or target sequence
  • the target site or target sequence bound and/or cleaved by a nuclease variant is double-stranded and comprises the reference sequence and its complement.
  • the target site is a sequence in a human IL-lORa gene.
  • Recombination refers to a process of exchange of genetic information between two polynucleotides, including but not limited to, donor capture by non-homologous end joining (HEJ) and homologous recombination.
  • HEJ non-homologous end joining
  • HR homologous recombination
  • HDR homology-directed repair
  • This process requires nucleotide sequence homology, uses a "donor” molecule as a template to repair a "target” molecule (i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target.
  • transfer can 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 is used to resynthesize genetic information that will become part of the target, and/or related processes.
  • Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.
  • NHEJ non-homologous end joining
  • cNHEJ The classical NHEJ pathway (cNHEJ) requires the KU/DNA-PKcs/Lig4/XRCC4 complex, ligates ends back together with minimal processing and often leads to precise repair of the break.
  • Alternative NHEJ pathways cNHEJ
  • exonucleases e.g., Trex2
  • Trex2 may bias repair towards an altNHEJ pathway.
  • “Cleavage” refers to the breakage of the covalent backbone of a DNA molecule.
  • Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double- stranded cleavage are possible. Double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends.
  • polypeptides and nuclease variants e.g., homing endonuclease variants, megaTALs, etc. contemplated herein are used for targeted double-stranded DNA cleavage. Endonuclease cleavage recognition sites may be on either DNA strand.
  • exogenous molecule is a molecule that is not normally present in a cell, but that is introduced into a cell by one or more genetic, biochemical or other methods.
  • exogenous molecules include, but are not limited to small organic molecules, protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.
  • lipid-mediated transfer i.e., liposomes, including neutral and cationic lipids
  • electroporation direct injection, cell fusion, particle bombardment, biopolymer nanoparticle, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.
  • An "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. Additional endogenous molecules can include proteins.
  • a “gene,” refers to a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences.
  • a gene includes, but is not limited to, promoter sequences, enhancers, silencers, insulators, boundary elements, terminators, polyadenylation sequences, post-transcription response elements, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, replication origins, matrix attachment sites, and locus control regions.
  • Gene expression refers to the conversion of the information, contained in a gene, into a gene product.
  • a gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA.
  • Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
  • genetically engineered or “genetically modified” refers to the chromosomal or extrachromosomal addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. Genetic modifications may be targeted or non- targeted to a particular site in a cell ' s genome. In one embodiment, genetic modification is site specific. In one embodiment, genetic modification is not site specific.
  • Genome editing refers to the substitution, deletion, and/or introduction of genetic material at a target site in the cell ' s genome, which restores, corrects, disrupts, and/or modifies expression of a gene or gene product.
  • Genome editing contemplated in particular embodiments comprises introducing one or more nuclease variants into a cell to generate DNA lesions at or proximal to a target site in the cell ' s genome, optionally in the presence of a donor repair template.
  • gene therapy refers to the introduction of extra genetic material into the total genetic material in a cell that restores, corrects, or modifies expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide.
  • introduction of genetic material into the cell ' s genome by genome editing that restores, corrects, disrupts, or modifies expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide is considered gene therapy.
  • immune disorder refers to a disease that evokes a response from the immune system.
  • the term “immune disorder” refers to a cancer, graft- versus-host disease, an autoimmune disease, or an immunodeficiency.
  • immune disorders encompasses infectious disease.
  • cancer relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues.
  • malignant refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood).
  • metastasis i.e., spread to other locations in the body via lymph or blood.
  • metastasize refers to the spread of cancer from one part of the body to another.
  • a tumor formed by cells that have spread is called a "metastatic tumor” or a “metastasis.”
  • the metastatic tumor contains cells that are like those in the original (primary) tumor.
  • Benign or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
  • a “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue.
  • a tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancers form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
  • the amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor.
  • graft- versus-host disease refers complications that can occur after cell, tissue, or solid organ transplant.
  • GVHD can occur after a stem cell or bone marrow transplant in which the transplanted donor cells attack the transplant recipient's body.
  • Acute GVHD in humans takes place within about 60 days post-transplantation and results in damage to the skin, liver, and gut by the action of cytolytic lymphocytes.
  • Chronic GVHD occurs later and is a systemic autoimmune disease that affects primarily the skin, resulting in the polyclonal activation of B cells and the hyperproduction of Ig and autoantibodies.
  • Solid-organ transplant graft-versus-host disease SOT-GVHD
  • the more common type is antibody mediated, wherein antibodies from a donor with blood type O attack a recipient's red blood cells in recipients with blood type A, B, or AB, leading to mild transient, hemolytic anemias.
  • the second form of SOT-GVHD is a cellular type associated with high mortality, wherein donor-derived T cells produce an immunological attack against immunologically disparate host tissue, most often in the skin, liver, gastrointestinal tract, and bone marrow, leading to complications in these organs.
  • "Graft- versus-leukemia” or "GVL” refer to an immune response to a person's leukemia cells by immune cells present in a donor's transplanted tissue, such as bone marrow or peripheral blood.
  • autoimmune disease refers to a disease in which the body produces an immunogenic (i.e., immune system) response to some constituent of its own tissue.
  • immune system i.e., immune system
  • the immune system loses its ability to recognize some tissue or system within the body as "self and targets and attacks it as if it were foreign.
  • autoimmune diseases include, but are not limited to: arthritis, inflammatory bowel disease, Hashimoto's thyroiditis, Grave's disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti -immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like.
  • Immunodeficiency means the state of a patient whose immune system has been compromised by disease or by administration of chemicals. This condition makes the system deficient in the number and type of blood cells needed to defend against a foreign substance.
  • Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immunodeficiency syndrome), SCID (severe combined immunodeficiency disease), selective IgA deficiency, common variable immunodeficiency, X-linked agammaglobulinemia, chronic granulomatous disease, hyper-IgM syndrome, Wiskott-Aldrich Syndrome (WAS), and diabetes.
  • infectious disease refers to a disease that can be transmitted from person to person or from organism to organism, and is caused by a microbial or viral agent (e.g., common cold). Infectious diseases are known in the art and include, for example, hepatitis, sexually transmitted diseases (e.g., Chlamydia, gonorrhea), tuberculosis, HIV/AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.
  • a microbial or viral agent e.g., common cold.
  • Infectious diseases include, for example, hepatitis, sexually transmitted diseases (e.g., Chlamydia, gonorrhea), tuberculosis, HIV/AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.
  • the terms "individual” and “subject” are often used interchangeably and refer to any animal that exhibits a symptom of an immune disorder that can be treated with the nuclease variants, genome editing compositions, gene therapy vectors, genome editing vectors, genome edited cells, and methods contemplated elsewhere herein.
  • Suitable subjects e.g., patients
  • laboratory animals such as mouse, rat, rabbit, or guinea pig
  • farm animals such as a cat or dog
  • domestic animals or pets such as a cat or dog.
  • Non-human primates and, preferably, human subjects are included.
  • Typical subjects include human patients that have, have been diagnosed with, or are at risk of having an immune disorder.
  • the term "patient” refers to a subject that has been diagnosed with an immune disorder that can be treated with the nuclease variants, genome editing compositions, gene therapy vectors, genome editing vectors, genome edited cells, and methods contemplated elsewhere herein.
  • treatment includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. Treatment can optionally involve delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.
  • prevention and similar words such as “prevention,” “prevented,” “preventing” etc. , indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition, e.g., cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.
  • the phrase "ameliorating at least one symptom of refers to decreasing one or more symptoms of the disease or condition for which the subject is being treated, e.g., cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and
  • the disease or condition being treated is a cancer
  • the one or more symptoms ameliorated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, distended or painful abdomen (due to enlarged abdominal organs), bone or joint pain, fractures, unplanned weight loss, poor appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function).
  • the term “amount” refers to "an amount effective” or “an effective amount” of a nuclease variant, genome editing composition, or genome edited cell sufficient to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.
  • prophylactically effective amount refers to an amount of a nuclease variant, genome editing composition, or genome edited cell sufficient to achieve the desired
  • prophylactic result typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.
  • composition, or genome edited cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects.
  • the term "therapeutically effective amount” includes an amount that is effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).
  • Nuclease variants contemplated in particular embodiments herein are suitable for genome editing a target site in the IL-lORa gene and comprise one or more DNA binding domains and one or more DNA cleavage domains (e.g., one or more endonuclease and/or exonuclease domains), and optionally, one or more linkers contemplated herein.
  • the terms "reprogrammed nuclease,” “engineered nuclease,” or “nuclease variant” are used
  • a nuclease variant binds and cleaves a target sequence in exon 2 of an IL-lORa gene, preferably at SEQ ID NO: 13 in exon 2 of an IL-lORa gene, and more preferably at the sequence "ATTC” in SEQ ID NO: 13 in exon 2 of an IL-lORa gene.
  • the nuclease variant may be designed and/or modified from a naturally occurring nuclease or from a previous nuclease variant.
  • Nuclease variants contemplated in particular embodiments may further comprise one or more additional functional domains, e.g., an end- processing enzymatic domain of an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' - 3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g., Trex2), 5 ' flap endonuclease, helicase, template-dependent DNA polymerases or template-independent DNA polymerase activity.
  • additional functional domains e.g., an end- processing enzymatic domain of an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' - 3 ' alkaline exonuclease, 3 ' -5 ' ex
  • nuclease variants that bind and cleave a target sequence in the
  • IL-lORa gene include, but are not limited to homing endonuclease (meganuclease) variants and megaTALs.
  • a homing endonuclease or meganuclease is reprogrammed to introduce a double-strand break (DSB) in a target site in an IL-lORa gene.
  • a homing endonuclease variant introduces a double strand break in exon 2 of an IL-lORa gene, preferably at SEQ ID NO: 13 in exon 2 of an IL-lORa gene, and more preferably at the sequence "ATTC" in SEQ ID NO: 13 in exon 2 of an IL-lORa gene.
  • Homing endonuclease and “meganuclease” are used interchangeably and refer to naturally- occurring homing endonucl eases that recognize 12-45 base-pair cleavage sites and are commonly grouped into five families based on sequence and structure motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D/E)XK.
  • a “reference homing endonuclease” or “reference meganuclease” refers to a wild type homing endonuclease or a homing endonuclease found in nature.
  • a “reference homing endonuclease” refers to a wild type homing endonuclease that has been modified to increase basal activity.
  • an “engineered homing endonuclease,” “reprogrammed homing endonuclease,” “homing endonuclease variant,” “engineered meganuclease,” “reprogrammed meganuclease,” or “meganuclease variant” refers to a homing endonuclease comprising one or more DNA binding domains and one or more DNA cleavage domains, wherein the homing endonuclease has been designed and/or modified from a parental or naturally occurring homing
  • the homing endonuclease variant may be designed and/or modified from a naturally occurring homing endonuclease or from another homing endonuclease variant.
  • Homing endonuclease variants contemplated in particular embodiments may further comprise one or more additional functional domains, e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g., Trex2), 5 ' flap endonuclease, helicase, template dependent DNA polymerase or template-independent DNA polymerase activity.
  • additional functional domains e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g., Trex2), 5 ' flap endonuclease, helicase, template dependent DNA
  • HE variants do not exist in nature and can be obtained by recombinant DNA technology or by random mutagenesis.
  • HE variants may be obtained by making one or more amino acid alterations, e.g., mutating, substituting, adding, or deleting one or more amino acids, in a naturally occurring HE or HE variant.
  • a HE variant comprises one or more amino acid alterations to the DNA recognition interface.
  • HE variants contemplated in particular embodiments may further comprise one or more linkers and/or additional functional domains, e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g., Trex2), 5 ' flap endonuclease, helicase, template-dependent DNA
  • HE variants are introduced into a T cell with an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g., Trex2), 5 ' flap endonuclease, helicase, template-dependent DNA polymerase or template-independent DNA polymerase activity.
  • an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g., Trex2), 5 ' flap endonuclease, helicase, template-dependent DNA polymerase or template-independent DNA polymerase activity.
  • the HE variant and 3 ' processing enzyme may be introduced separately, e.g., in different vectors or separate mRNAs, or together, e.g., as a fusion protein, or in a polycistronic construct separated by a viral self-cleaving peptide or an IRES element.
  • a "DNA recognition interface” refers to the HE amino acid residues that interact with nucleic acid target bases as well as those residues that are adjacent.
  • the DNA recognition interface comprises an extensive network of side chain-to-side chain and side chain-to-DNA contacts, most of which is necessarily unique to recognize a particular nucleic acid target sequence.
  • the amino acid sequence of the DNA recognition interface corresponding to a particular nucleic acid sequence varies significantly and is a feature of any natural or HE variant.
  • a HE variant contemplated in particular embodiments may be derived by constructing libraries of HE variants in which one or more amino acid residues localized in the DNA recognition interface of the natural HE (or a previously generated HE variant) are varied.
  • the libraries may be screened for target cleavage activity against each predicted IL-lORa target site using cleavage assays (see e.g., Jarjour et al, 2009. Nuc. Acids Res. 37(20): 6871-6880).
  • LAGLIDADG homing endonucleases are the most well studied family of homing endonucleases, are primarily encoded in archaea and in organellar DNA in green algae and fungi, and display the highest overall DNA recognition specificity. LHEs comprise one or two LAGLIDADG catalytic motifs per protein chain and function as homodimers or single chain monomers, respectively. Structural studies of LAGLIDADG proteins identified a highly conserved core structure (Stoddard 2005), characterized by an ⁇ fold, with the
  • LAGLIDADG motif belonging to the first helix of this fold belongs to the first helix of this fold.
  • the highly efficient and specific cleavage of LHE ' s represent a protein scaffold to derive novel, highly specific endonucleases.
  • engineering LHEs to bind and cleave a non-natural or non-canonical target site requires selection of the appropriate LHE scaffold, examination of the target locus, selection of putative target sites, and extensive alteration of the LHE to alter its DNA contact points and cleavage specificity, at up to two-thirds of the base-pair positions in a target site.
  • LHEs from which reprogrammed LHEs or LHE variants may be designed include, but are not limited to I-Crel and I-Scel.
  • LHEs from which reprogrammed LHEs or LHE variants may be designed include, but are not limited to I-AabMI, I-AaeMI, I-Anil, I-ApaMI, I-CapIII, I- CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I- EjeMI, I-GpeMI, I-Gpil, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-Ltrll, I-Ltrl, I-LtrWI, I- MpeMI, I-MveMI, I-Ncrll, I-Ncrl, I-NcrMI, I-OheMI, I-Onul, I-OsoMI, I-OsoMII, I-OsoMII
  • the reprogrammed LHE or LHE variant is selected from the group consisting of: an I-CpaMI variant, an I-HjeMI variant, an I-Onul variant, an I-PanMI variant, and an I-SmaMI variant.
  • the reprogrammed LHE or LHE variant is an I-Onul variant. See e.g., SEQ ID NOs: 6-8.
  • reprogrammed I-Onul LHEs or I-Onul variants targeting the IL- 10Ra gene were generated from a natural I-Onul or biologically active fragment thereof (SEQ ID NOs: 1-5).
  • reprogrammed I-Onul LHEs or I-Onul variants targeting the human IL-lORa gene were generated from an existing I-Onul variant.
  • reprogrammed I-Onul LHEs were generated against a human IL- 1 ORa gene target site set forth in SEQ ID NO: 13.
  • the reprogrammed I-Onul LHE or I-Onul variant that binds and cleaves a human IL-lORa gene comprises one or more amino acid substitutions in the DNA recognition interface.
  • the I-Onul LHE that binds and cleaves a human IL-lORa gene comprises at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the DNA recognition interface of I-Onul (Taekuchi e
  • the I-Onul LHE that binds and cleaves a human IL-lORa gene comprises at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 99% sequence identity with the DNA recognition interface of I-Onul (Taekuchi et al. 2011. Proc Natl Acad Sci U. S. A. 2011 Aug 9; 108(32): 13077-13082) or an l- Onul LHE variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
  • an I-Onul LHE variant that binds and cleaves a human IL- lORa gene comprises one or more amino acid substitutions or modifications in the DNA recognition interface of an I-Onul as set forth in any one of SEQ ID NOs: 1-8.
  • an I-Onul LHE variant that binds and cleaves a human IL- 10Ra gene comprises one or more amino acid substitutions or modifications in the DNA recognition interface, particularly in the subdomains situated from positions 24-50, 68 to 82, 180 to 203 and 223 to 240 of I-Onul (SEQ ID NOs: 1-5) or an I-Onul variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
  • an I-Onul LHE that binds and cleaves a human IL- 1 ORa gene comprises one or more amino acid substitutions or modifications in the DNA recognition interface at amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of I-Onul (SEQ ID NOs: 1-5) or an I-Onul variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
  • an I-Onul LHE variant that binds and cleaves a human IL- 10Ra gene comprises 5, 10, 15, 20, 25, 30, 35, or 40 or more amino acid substitutions or modifications in the DNA recognition interface, particularly in the subdomains situated from positions 24-50, 68 to 82, 180 to 203 and 223 to 240 of I-Onul (SEQ ID NOs: 1-5) or an I- Onul variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
  • an I-Onul LHE variant that binds and cleaves a human IL- 10Ra gene comprises 5, 10, 15, 20, 25, 30, 35, or 40 or more amino acid substitutions or modifications in the DNA recognition interface at amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of (I-Onul SEQ ID NOs: 1-5) or an I- Onul variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
  • an I-Onul LHE variant that binds and cleaves a human IL-lORa gene comprises one or more amino acid substitutions or modifications at additional positions situated anywhere within the entire I-Onul sequence.
  • the residues which may be substituted and/or modified include but are not limited to amino acids that contact the nucleic acid target or that interact with the nucleic acid backbone or with the nucleotide bases, directly or via a water molecule.
  • an I-Onul LHE variant contemplated herein that binds and cleaves a human IL-lORa gene comprises one or more substitutions and/or modifications, preferably at least 5, preferably at least 10, preferably at least 15, preferably at least 20, more preferably at least 25, more preferably at least 30, even more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions in at least one position selected from the position group consisting of positions: 24, 26, 28, 30, 32, 34, 36, 37, 38, 40, 41, 42, 44, 46, 48, 59, 70, 72, 75, 78, 80, 82, 138, 143, 145, 159, 168, 180, 182, 184, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 207, 223, 225, 227, 228, 229, 232, 236, 238, and 240, of I-Onul (SEQ ID NOs: 1-5)
  • an I-Onul LHE variant that binds and cleaves a human IL- lORa gene comprises at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46V, Q46I, T48G, N59S, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180H, C180Y, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227
  • an I-Onul LHE variant that binds and cleaves a human IL-lORa gene comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46V, T48G, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, S159P, F168L, C180H, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, K229A, F232K, D236K, and V238I or an I-Onul variant as set forth in any one of SEQ ID NOs: 6-8,
  • an I-Onul LHE variant that binds and cleaves a human IL-lORa gene comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46I, T48G, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180H, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K, and V238I, or an I-Onul variant as set forth in any
  • an I-Onul LHE variant that binds and cleaves a human IL-lORa gene comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46I, T48G, N59S, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180Y, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K, and V238I, or an I-Onul variant as
  • an I-Onul LHE variant that binds and cleaves a human IL- 10Ra gene comprises an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 6-8, or a biologically active fragment thereof.
  • an I-Onul LHE variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 6-8, or a biologically active fragment thereof.
  • an I-Onul LHE variant comprises an amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.
  • an I-Onul LHE variant comprises an amino acid sequence set forth in SEQ ID NO: 7, or a biologically active fragment thereof.
  • an I-Onul LHE variant comprises an amino acid sequence set forth in SEQ ID NO: 8, or a biologically active fragment thereof.
  • a megaTAL comprising a homing endonuclease variant is reprogrammed to introduce a double-strand break (DSB) in a target site in an IL-lORa gene.
  • a homing endonuclease variant is reprogrammed to introduce a DSB in a target site in exon 2 of an IL-lORa gene, preferably at SEQ ID NO: 13 in exon 2 of an IL- 10Ra gene, and more preferably at the sequence "ATTC" in SEQ ID NO: 13 in exon 2 of an IL-lORa gene.
  • a "megaTAL” refers to a polypeptide comprising a TALE DNA binding domain and a homing endonuclease variant that binds and cleaves a DNA target sequence in an IL-lORa gene, and optionally comprises one or more linkers and/or additional functional domains, e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 '-3' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g., Trex2), 5 ' flap endonuclease, helicase or template-independent DNA polymerase activity.
  • end-processing enzymatic domain of an end-processing enzyme that exhibits 5 '-3' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g
  • a megaTAL can be introduced into a cell along with an end- processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g., Trex2), 5 ' flap endonuclease, helicase, template-dependent DNA
  • the megaTAL and 3 ' processing enzyme may be introduced separately, e.g., in different vectors or separate mRNAs, or together, e.g., as a fusion protein, or in a polycistronic construct separated by a viral self- cleaving peptide or an IRES element.
  • TALE DNA binding domain is the DNA binding portion of transcription activator-like effectors (TALE or TAL-effectors), which mimics plant transcriptional activators to manipulate the plant transcriptome (see e.g., Kay etal., 2007. Science 318:648-651).
  • TALE DNA binding domains contemplated in particular embodiments are engineered de novo or from naturally occurring TALEs, e.g., AvrBs3 from Xanthomonas campestris pv.
  • vesicatoria Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonas euvesicatoria, and
  • TALE proteins for deriving and designing DNA binding domains are disclosed in U.S. Patent No. 9,017,967, and references cited therein, all of which are incorporated herein by reference in their entireties.
  • a megaTAL comprises a TALE DNA binding domain comprising one or more repeat units that are involved in binding of the TALE DNA binding domain to its corresponding target DNA sequence.
  • a single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length.
  • Each TALE DNA binding domain repeat unit includes 1 or 2 DNA-binding residues making up the Repeat Variable Di-Residue (RVD), typically at positions 12 and/or 13 of the repeat.
  • RVD Repeat Variable Di-Residue
  • the natural (canonical) code for DNA recognition of these TALE DNA binding domains has been determined such that an HD sequence at positions 12 and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, NN binds to G or A, and NG binds to T.
  • C cytosine
  • NG binds to T
  • NI to A NI to A
  • NN binds to G or A
  • NG binds to T.
  • non-canonical (atypical) RVDs are contemplated.
  • Illustrative examples of non-canonical RVDs suitable for use in particular megaTALs contemplated in particular embodiments include, but are not limited to HH, KH, NH, NK, NQ, RH, RN, SS, NN, SN, KN for recognition of guanine (G); NI, KI, RI, HI, SI for recognition of adenine (A); NG, HG, KG, RG for recognition of thymine (T); RD, SD, HD, ND, KD, YG for recognition of cytosine (C); NV, HN for recognition of A or G; and H*, HA, KA, N*, NA, NC, NS, RA, S*for recognition of A or T or G or C, wherein (*) means that the amino acid at position 13 is absent. Additional illustrative examples of RVDs suitable for use in particular megaTALs contemplated in particular embodiments further include those disclosed in U. S. Patent No. 8,614,09
  • a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 3 to 30 repeat units.
  • a megaTAL comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 TALE DNA binding domain repeat units.
  • a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 5-15 repeat units, more preferably 7-15 repeat units, more preferably 9-15 repeat units, and more preferably 9, 10, 11, 12, 13, 14, or 15 repeat units.
  • a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 3 to 30 repeat units and an additional single truncated TALE repeat unit comprising 20 amino acids located at the C-terminus of a set of TALE repeat units, i.e., an additional C-terminal half-TALE DNA binding domain repeat unit (amino acids -20 to -1 of the C-cap disclosed elsewhere herein, infra).
  • a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 3.5 to 30.5 repeat units.
  • a megaTAL comprises 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, 15.5, 16.5, 17.5, 18.5, 19.5, 20.5, 21.5, 22.5, 23.5, 24.5, 25.5, 26.5, 27.5, 28.5, 29.5, or 30.5 TALE DNA binding domain repeat units.
  • a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 5.5-15.5 repeat units, more preferably 7.5-15.5 repeat units, more preferably 9.5-15.5 repeat units, and more preferably 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, or 15.5 repeat units.
  • a megaTAL comprises a TAL effector architecture comprising an "N-terminal domain (NTD)" polypeptide, one or more TALE repeat domains/units, a "C-terminal domain (CTD)” polypeptide, and a homing endonuclease variant.
  • NTD N-terminal domain
  • CTD C-terminal domain
  • the NTD, TALE repeats, and/or CTD domains are from the same species.
  • one or more of the NTD, TALE repeats, and/or CTD domains are from different species.
  • NTD N-terminal domain
  • the NTD sequence if present, may be of any length as long as the TALE DNA binding domain repeat units retain the ability to bind DNA.
  • the NTD polypeptide comprises at least 120 to at least 140 or more amino acids N-terminal to the TALE DNA binding domain (0 is amino acid 1 of the most N-terminal repeat unit).
  • the NTD polypeptide comprises at least about 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or at least 140 amino acids N-terminal to the TALE DNA binding domain.
  • a megaTAL contemplated herein comprises an NTD polypeptide of at least about amino acids +1 to +122 to at least about +1 to +137 of aXanthomonas TALE protein (0 is amino acid 1 of the most N- terminal repeat unit).
  • the NTD polypeptide comprises at least about 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137 amino acids N-terminal to the TALE DNA binding domain of aXanthomonas TALE protein.
  • a megaTAL contemplated herein comprises an NTD polypeptide of at least amino acids +1 to +121 of aRalstonia TALE protein (0 is amino acid 1 of the most N-terminal repeat unit).
  • the NTD polypeptide comprises at least about 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137 amino acids N-terminal to the TALE DNA binding domain of a Ralstonia TALE protein.
  • CTD C-terminal domain
  • the CTD sequence if present, may be of any length as long as the TALE DNA binding domain repeat units retain the ability to bind DNA.
  • the CTD polypeptide comprises at least 20 to at least 85 or more amino acids C-terminal to the last full repeat of the TALE DNA binding domain (the first 20 amino acids are the half-repeat unit C-terminal to the last C-terminal full repeat unit).
  • the CTD polypeptide comprises at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 443, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 76, 77, 78, 79, 80, 81, 82, 83, 84, or at least 85 amino acids C-terminal to the last full repeat of the TALE DNA binding domain.
  • a megaTAL contemplated herein comprises a CTD polypeptide of at least about amino acids -20 to -1 of a Xanthomonas TALE protein (-20 is amino acid 1 of a half- repeat unit C-terminal to the last C-terminal full repeat unit).
  • the CTD polypeptide comprises at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids C-terminal to the last full repeat of the TALE DNA binding domain of a Xanthomonas TALE protein.
  • a megaTAL contemplated herein comprises a CTD polypeptide of at least about amino acids -20 to -1 of aRalstonia TALE protein (-20 is amino acid 1 of a half-repeat unit C-terminal to the last C-terminal full repeat unit).
  • the CTD polypeptide comprises at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids C-terminal to the last full repeat of the TALE DNA binding domain of a Ralstonia TALE protein.
  • a megaTAL contemplated herein comprises a fusion polypeptide comprising a TALE DNA binding domain engineered to bind a target sequence, a homing endonuclease reprogrammed to bind and cleave a target sequence, and optionally an NTD and/or CTD polypeptide, optionally joined to each other with one or more linker polypeptides contemplated elsewhere herein.
  • a megaTAL comprising TALE DNA binding domain, and optionally an NTD and/or CTD polypeptide is fused to a linker polypeptide which is further fused to a homing endonuclease variant.
  • the TALE DNA binding domain binds a DNA target sequence that is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides away from the target sequence bound by the DNA binding domain of the homing endonuclease variant.
  • the megaTALs contemplated herein increase the specificity and efficiency of genome editing.
  • a megaTAL comprises a homing endonuclease variant and a
  • TALE DNA binding domain that binds a nucleotide sequence that is within about 4, 5, or 6 nucleotides, preferably, 6 nucleotides upstream of the binding site of the reprogrammed homing endonuclease.
  • a megaTAL comprises a homing endonuclease variant and a TALE DNA binding domain that binds the nucleotide sequence set forth in SEQ ID NO: 14, which is 6 nucleotides upstream of the nucleotide sequence bound and cleaved by the homing endonuclease variant (SEQ ID NO: 13).
  • the megaTAL target sequence is SEQ ID NO: 15.
  • a megaTAL contemplated herein comprises one or more TALE DNA binding repeat units and an LHE variant designed or reprogrammed from an LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMTV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I- GpeMI, I-Gpil, I-GzeMI, I-GzeMII, I-GzeMin, I-HjeMI, I-Ltrll, I-Ltrl, I-LtrWI, I-MpeMI, I- MveMI, I-Ncrll, I-Ncrl, I-NcrMI, I-OheMI, I-Onul, I-O
  • a megaTAL contemplated herein comprises an NTD, one or more TALE DNA binding repeat units, a CTD, and an LHE variant selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, I-ApaMI, I-CapHI, I-CapIV, I-CkaMI, I-CpaMI, I- CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-Gpil, I- GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I- Ncrl, I-NcrMI, I-OheMI, I-Onul, I-I
  • a megaTAL contemplated herein comprises an NTD, about 9.5 to about 15.5 TALE DNA binding repeat units, and an LHE variant selected from the group consisting of: I-AabMI, I-AaeMI, I- Anil, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I- CpaMI, I-CpaMII, I-CpaMHI, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-Ej eMI, I-GpeMI, I- Gpil, I-GzeMI, I-GzeMII, I-GzeMHI, I-HjeMI, I-LtrII, I-Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I- Ncrll, I-Ncrl, I-NcrMI, I-OheMI, I-Onul, I-
  • a megaTAL contemplated herein comprises an NTD of about 122 amino acids to 137 amino acids, about 9.5, about 10.5, about 11.5, about 12.5, about 13.5, about 14.5, or about 15.5 binding repeat units, a CTD of about 20 amino acids to about 85 amino acids, and an I-Onul LHE variant.
  • any one of, two of, or all of the NTD, DNA binding domain, and CTD can be designed from the same species or different species, in any suitable combination.
  • a megaTAL contemplated herein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-11.
  • a megaTAL-Trex2 fusion protein contemplated herein comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • a megaTAL comprises a TALE DNA binding domain and an I-Onul LHE variant binds and cleaves the nucleotide sequence set forth in SEQ ID NO: 15.
  • Genome editing compositions and methods contemplated in particular embodiments comprise editing cellular genomes using a nuclease variant and an end-processing enzyme.
  • a single polynucleotide encodes a homing endonuclease variant and an end-processing enzyme, separated by a linker, a self cleaving peptide sequence, e.g., 2A sequence, or by an IRES sequence.
  • genome editing compositions comprise a polynucleotide encoding a nuclease variant and a separate polynucleotide encoding an end-processing enzyme.
  • end-processing enzyme refers to an enzyme that modifies the exposed ends of a polynucleotide chain.
  • the polynucleotide may be double-stranded DNA (dsDNA), single- stranded DNA (ssDNA), RNA, double-stranded hybrids of DNA and RNA, and synthetic DNA (for example, containing bases other than A, C, G, and T).
  • An end-processing enzyme may modify exposed polynucleotide chain ends by adding one or more nucleotides, removing one or more nucleotides, removing or modifying a phosphate group and/or removing or modifying a hydroxyl group.
  • An end-processing enzyme may modify ends at endonuclease cut sites or at ends generated by other chemical or mechanical means, such as shearing (for example by passing through fine-gauge needle, heating, sonicating, mini bead tumbling, and nebulizing), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolysis and chemotherapy agents.
  • genome editing compositions and methods contemplated in particular embodiments comprise editing cellular genomes using a homing endonuclease variant or megaTAL and a DNA end-processing enzyme.
  • DNA end-processing enzyme refers to an enzyme that modifies the exposed ends of DNA.
  • a DNA end-processing enzyme may modify blunt ends or staggered ends (ends with 5 ' or 3 ' overhangs).
  • a DNA end-processing enzyme may modify single stranded or double stranded DNA.
  • a DNA end-processing enzyme may modify ends at endonuclease cut sites or at ends generated by other chemical or mechanical means, such as shearing (for example by passing through fine-gauge needle, heating, sonicating, mini bead tumbling, and nebulizing), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolysis and chemotherapy agents.
  • DNA end-processing enzyme may modify exposed DNA ends by adding one or more nucleotides, removing one or more nucleotides, removing or modifying a phosphate group and/or removing or modifying a hydroxyl group.
  • Illustrative examples of DNA end-processing enzymes suitable for use in particular embodiments contemplated herein include, but are not limited to: 5 ' -3 ' exonucleases, 5 ' -3 ' alkaline exonucleases, 3 ' -5 ' exonucleases, 5 ' flap endonucleases, helicases, phosphatases, hydrolases and template-independent DNA polymerases.
  • DNA end-processing enzymes suitable for use in particular embodiments contemplated herein include, but are not limited to, Trex2, Trexl, Trexl without transmembrane domain, Apollo, Artemis, DNA2, Exol, ExoT, ⁇ , Fenl, Fanl, Mrell, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, Lambda exonuclease, Sox, Vaccinia DNA polymerase, exonuclease I, exonuclease III, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7-exonuclease Gene 6, avian myeloblastosis virus integration protein (IN), Bloom, Antartic Phophatase, Alkaline
  • Phosphatase Poly nucleotide Kinase (PNK), Apel, Mung Bean nuclease, Hexl, TTRAP (TDP2), Sgsl, Sae2, CUP, Pol mu, Pol lambda, MUS81, EMEl, EME2, SLXl, SLX4 and UL- 12.
  • genome editing compositions and methods for editing cellular genomes contemplated herein comprise polypeptides comprising a homing
  • exonuclease variant or megaTAL refers to enzymes that cleave phosphodiester bonds at the end of a polynucleotide chain via a hydrolyzing reaction that breaks phosphodiester bonds at either the 3 ' or 5 ' end.
  • exonucleases suitable for use in particular embodiments contemplated herein include, but are not limited to: hExoI, Yeast Exol, E. coli Exol, hTREX2, mouse TREX2, rat TREX2, hTREXl, mouse TREXl, rat TREX1, and Rat TREXl.
  • the DNA end-processing enzyme is a 3' or 5' exonuclease, preferably Trex 1 or Trex2, more preferably Trex2, and even more preferably human or mouse Trex2.
  • Nuclease variants contemplated in particular embodiments can be designed to bind to any suitable target sequence and can have a novel binding specificity, compared to a naturally- occurring nuclease.
  • the target site is a regulatory region of a gene including, but not limited to promoters, enhancers, repressor elements, and the like.
  • the target site is a coding region of a gene or a splice site.
  • nuclease variants are designed to down-regulate or decrease expression of a gene.
  • a nuclease variant and donor repair template can be designed to repair or delete a desired target sequence.
  • nuclease variants bind to and cleave a target sequence in an interleukin 10 receptor 1 alpha (IL-lORa) gene.
  • IL-lORa is also referred to as CDW210A, IL-10R1, IL-10RA, CD210 Antigen, ML-IOR, CD210a, CD210, and IBD28.
  • the IL-lORa gene encodes a ⁇ 63kD protein that is expressed in the spleen, thymus, and PBMCs and is highly expressed in monocytes, B cells, large granular lymphocytes, and T cells.
  • IL-lORa is also weakly expressed in pancreas, skeletal muscle, brain, heart, and kidney tissues, and intermediately expressed in placenta, lung, and liver tissue.
  • IL-10 activates downstream signaling by binding to the IL-10 receptor (IL-IOR), comprised of two a subunits (encoded by IL-lORa) and two ⁇ subunits (encoded by IL-IORP).
  • IL-10 mediates immunosuppressive signals via the IL- 1 ORa by inhibiting proinflammatory cytokine synthesis.
  • Loss of IL- 1 ORa expression in regulatory T cells (Tregs) impairs the immune system's response to GVHD, inflammatory and autoimmune diseases.
  • a homing endonuclease variant or megaTAL introduces a double-strand break (DSB) in a target site in an IL-lORa gene.
  • DSB double-strand break
  • a homing endonuclease variant or megaTAL introduces a DSB in exon 2 of an IL-lORa gene, preferably at SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of an IL- lORa gene, and more preferably at the sequence "ATTC” in SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of an IL-lORa gene.
  • a homing endonuclease variant or megaTAL cleaves double-stranded DNA and introduces a DSB into the polynucleotide sequence set forth in SEQ ID NO: 13 or 15.
  • the IL-lORa gene is a human IL-lORa gene.
  • Nuclease variants may be used to introduce a DSB in a target sequence; the DSB may be repaired through homology directed repair (HDR) mechanisms in the presence of one or more donor repair templates.
  • the donor repair template is used to insert a sequence into the genome.
  • the donor repair template is used to repair or modify a sequence in the genome.
  • a donor repair template is introduced into a hematopoietic cell, e.g., a T cell, by transducing the cell with an adeno-associated virus (AAV), retrovirus, e.g., lentivirus, IDLV, etc., herpes simplex virus, adenovirus, or vaccinia virus vector comprising the donor repair template.
  • AAV adeno-associated virus
  • retrovirus e.g., lentivirus, IDLV, etc.
  • herpes simplex virus e.g., lentivirus, IDLV, etc.
  • adenovirus e.g., lentivirus, IDLV, etc.
  • herpes simplex virus e.g., lentivirus, IDLV, etc.
  • vaccinia virus vector comprising the donor repair template.
  • the donor repair template comprises one or more homology arms that flank the DSB site.
  • the term “homology arms” refers to a nucleic acid sequence in a donor repair template that is identical, or nearly identical, to DNA sequence flanking the DNA break introduced by the nuclease at a target site.
  • the donor repair template comprises a 5 ' homology arm that comprises a nucleic acid sequence that is identical or nearly identical to the DNA sequence 5 ' of the DNA break site.
  • the donor repair template comprises a 3 ' homology arm that comprises a nucleic acid sequence that is identical or nearly identical to the DNA sequence 3 ' of the DNA break site.
  • the donor repair template comprises a 5 ' homology arm and a 3 ' homology arm.
  • the donor repair template may comprise homology to the genome sequence immediately adjacent to the DSB site, or homology to the genomic sequence within any number of base pairs from the DSB site.
  • the donor repair template comprises a nucleic acid sequence that is homologous to a genomic sequence about 5 bp, about 10 bp, about 25 bp, about 50 bp, about 100 bp, about 250 bp, about 500 bp, about 1000 bp, about 2500 bp, about 5000 bp, about 10000 bp or more, including any intervening length of homologous sequence.
  • suitable lengths of homology arms may be independently selected, and include but are not limited to: about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, about 1500 bp, about 1600 bp, about 1700 bp, about 1800 bp, about 1900 bp, about 2000 bp, about 2100 bp, about 2200 bp, about 2300 bp, about 2400 bp, about 2500 bp, about 2600 bp, about 2700 bp, about 2800 bp, about 2900 bp, or about 3000 bp, or longer homology arms, including all intervening lengths of homology arms.
  • suitable homology arm lengths include, but are not limited to: about 100 bp to about 3000 bp, about 200 bp to about 3000 bp, about 300 bp to about 3000 bp, about 400 bp to about 3000 bp, about 500 bp to about 3000 bp, about 500 bp to about 2500 bp, about 500 bp to about 2000 bp, about 750 bp to about 2000 bp, about 750 bp to about 1500 bp, or about 1000 bp to about 1500 bp, including all intervening lengths of homology arms.
  • the lengths of the 5 ' and 3 ' homology arms are independently selected from about 500 bp to about 1500 bp. In one embodiment, the
  • 5 ' homology arm is about 1500 bp and the 3 ' homology arm is about 1000 bp. In one embodiment, the 5 ' homology arm is between about 200 bp to about 600 bp and the 3 ' homology arm is between about 200 bp to about 600 bp. In one embodiment, the 5 ' homology arm is about 200 bp and the 3 ' homology arm is about 200 bp. In one embodiment, the 5 ' homology arm is about 300 bp and the 3 ' homology arm is about 300 bp. In one
  • the 5 ' homology arm is about 400 bp and the 3 ' homology arm is about 400 bp. In one embodiment, the 5 ' homology arm is about 500 bp and the 3 ' homology arm is about 500 bp. In one embodiment, the 5 ' homology arm is about 600 bp and the 3 ' homology arm is about 600 bp.
  • Donor repair templates may further comprises one or more polynucleotides such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Art sites), termination codons, transcriptional termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, contemplated elsewhere herein.
  • polynucleotides such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Art sites), termination codons, transcriptional termination signals, and polynucleot
  • the donor repair template comprises a 5 ' homology arm, an RNA polymerase II promoter, one or more polynucleotides encoding a therapeutic gene or fragment thereof, transgene or selectable marker, and a 3 ' homology arm.
  • a target site is modified with a donor repair template comprising a 5 ' homology arm, one or more polynucleotides encoding self-cleaving viral peptide, e.g., T2A, a therapeutic gene or fragment thereof, transgene or selectable marker, optionally a poly(A) signal, and a 3 ' homology arm.
  • the donor repair template comprises one or more
  • polynucleotides encoding a therapeutic gene or fragment thereof, transgene, or selectable marker.
  • the donor repair template comprises one or more
  • a bispecific T cell engager BiTE
  • a cytokine e.g., IL-2, insulin, IFN- ⁇ , IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-a
  • a chemokine e.g., ⁇ - ⁇ , ⁇ - ⁇ , MCP-1, MCP-3, and RANTES
  • a cytotoxin e.g., Perforin, Granzyme A, and Granzyme B
  • a cytokine receptor e.g., an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, and an IL-21 receptor
  • an engineered antigen receptor e.g., an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a Daric receptor or components thereof, or a chimeric cytokine receptor receptor.
  • engineered TCR refers to a T cell receptor, e.g., an ⁇ TCR that has a high-avidity and reactivity toward a target antigen.
  • the engineered TCR may be selected, cloned, and subsequently introduced into a population of T cells used for adoptive immunotherapy.
  • An engineered TCR is an exogenous TCR because it is introduced into T cells that do not normally express the particular TCR.
  • the essential aspect of the engineered TCRs is that it has high avidity for a tumor antigen presented by a major histocompatibility complex (MHC) or similar immunological component.
  • MHC major histocompatibility complex
  • CARs are engineered to bind target antigens in an MHC independent manner.
  • CAR refers to a chimeric antigen receptor.
  • Illustrative examples of CARs are disclosed in PCT Publication Nos. : WO2015164759, WO2015188119, and WO2016014789, each of which is incorporated herein by reference in its entirety.
  • Diagonal receptor refers to a multichain engineered antigen receptor.
  • Daric architectures and components thereof are disclosed in PCT Publication No. WO2015/017214 and U.S. Patent Publication No. 20150266973, each of which is incorporated herein by reference in its entirety.
  • chimeric cytokine receptor or "zetakine” refer to chimeric transmembrane immunoreceptors that comprise an extracellular domain comprising a soluble receptor ligand linked to a support region capable of tethering the extracellular domain to a cell surface, a transmembrane region and an intracellular signaling domain.
  • zetakines are disclosed in U.S. Patent Nos.: 7,514,537; 8,324,353; 8,497,118; and
  • the donor repair template comprises a polynucleotide comprising an IL-lORa gene or portion thereof and is designed to introduce one or more mutations in a genomic IL-lORa sequence such that a mutant IL-lORa gene product is expressed.
  • the donor repair template comprises a polynucleotide encoding FoxP3, a polypeptide that increases expression of FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
  • the donor repair template comprises a polynucleotide comprising an IL-lORa gene or portion thereof and is designed to correct one or more mutations in a genomic IL-lORa sequence such that a wild type IL-lORa gene product is expressed.
  • the donor template is designed such that a polynucleotide is inserted at a target site in the IL-lORa gene without substantially disrupting IL-lORa expression.
  • polypeptides are contemplated herein, including, but not limited to, homing endonuclease variants, megaTALs, and fusion polypeptides.
  • a polypeptide comprises the amino acid sequence set forth in SEQ ID NOs: 1-12 and 21.
  • Polypeptide “polypeptide fragment,” “peptide” and “protein” are used interchangeably, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids.
  • a "polypeptide” includes fusion polypeptides and other variants.
  • Polypeptides can be prepared using any of a variety of well-known recombinant and/or synthetic techniques. Polypeptides are not limited to a specific length, e.g., they may comprise a full length protein sequence, a fragment of a full length protein, or a fusion protein, and may include post-translational modifications of the polypeptide, for example,
  • glycosylations acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • isolated protein refers to in vitro synthesis, isolation, and/or purification of a peptide or polypeptide molecule from a cellular environment, and from association with other components of the cell, i.e., it is not significantly associated with in vivo substances.
  • polypeptides contemplated in particular embodiments include, but are not limited to homing endonuclease variants, megaTALs, end-processing nucleases, fusion polypeptides and variants thereof.
  • Polypeptides include "polypeptide variants.” Polypeptide variants may differ from a naturally occurring polypeptide in one or more amino acid substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more amino acids of the above polypeptide sequences. For example, in particular embodiments, it may be desirable to improve the biological properties of a homing endonuclease, megaTAL or the like that binds and cleaves a target site in the human IL-lORa gene by introducing one or more substitutions, deletions, additions and/or insertions into the polypeptide.
  • polypeptides include polypeptides having at least about 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) amino acid identity to any of the reference sequences contemplated herein, typically where the variant maintains at least one biological activity of the reference sequence.
  • Polypeptides variants include biologically active "polypeptide fragments.”
  • biologically active polypeptide fragments include DNA binding domains, nuclease domains, and the like.
  • biologically active fragment or minimal biologically active fragment refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%), at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity.
  • the biological activity is binding affinity and/or cleavage activity for a target sequence.
  • a polypeptide fragment can comprise an amino acid chain at least 5 to about 1700 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids long.
  • a polypeptide comprises a biologically active fragment of a homing endonuclease variant.
  • the polypeptides set forth herein may comprise one or more amino acids denoted as "X.”
  • "X" if present in an amino acid SEQ ID NO, refers to any amino acid.
  • One or more "X" residues may be present at the N- and C-terminus of an amino acid sequence set forth in particular SEQ ID NOs contemplated herein. If the "X' amino acids are not present the remaining amino acid sequence set forth in a SEQ ID NO may be considered a biologically active fragment.
  • a polypeptide comprises a biologically active fragment of a homing endonuclease variant, e.g., SEQ ID NOs: 3-5.
  • the biologically active fragment may comprise an N-terminal truncation and/or C-terminal truncation.
  • a biologically active fragment lacks or comprises a deletion of the 1, 2, 3, 4, 5, 6, 7, or 8 N- terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence, more preferably a deletion of the 4 N-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence.
  • a biologically active fragment lacks or comprises a deletion of the 1, 2, 3, 4, or 5 C-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence, more preferably a deletion of the 2 C-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence.
  • a biologically active fragment lacks or comprises a deletion of the 1, 2, 3, 4, or 5 C-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence, more preferably a deletion of the 2 C-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence.
  • a biologically active fragment lacks or comprises a deletion of the 4 N-terminal amino acids and 2 C-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence.
  • an I-Onul variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or a deletion of the following 1, 2, 3, 4, or 5 C-terminal amino acids: R, G, S, F, V.
  • an I-Onul variant comprises a deletion or substitution of 1, 2, 3, 4, 5, 6, 7, or 8 the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or a deletion or substitution of the following 1, 2, 3, 4, or 5 C-terminal amino acids: R, G, S, F, V.
  • an I-Onul variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or a deletion of the following 1 or 2 C-terminal amino acids: F, V.
  • an I-Onul variant comprises a deletion or substitution of 1,
  • N-terminal amino acids M, A, Y, M, S, R, R, E; and/or a deletion or substitution of the following 1 or 2 C-terminal amino acids: F, V.
  • polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367- 382), U.S. Pat. No. 4,873,192, Watson, J. D. et al, (Molecular Biology of the Gene, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif, 1987) and the references cited therein.
  • a variant will contain one or more conservative substitutions.
  • a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications may be made in the structure of the polynucleotides and polypeptides contemplated in particular embodiments, polypeptides include polypeptides having at least about and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.
  • amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids.
  • a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
  • Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non- polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
  • Suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule.
  • Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson etal. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p.224).
  • polynucleotide sequences encoding them can be separated by and IRES sequence as disclosed elsewhere herein.
  • Fusion polypeptides contemplated in particular embodiments include fusion polypeptides.
  • fusion polypeptides and polynucleotides encoding fusion polypeptides are provided.
  • Fusion polypeptides and fusion proteins refer to a polypeptide having at least two, three, four, five, six, seven, eight, nine, or ten polypeptide segments.
  • two or more polypeptides can be expressed as a fusion protein that comprises one or more self-cleaving polypeptide sequences as disclosed elsewhere herein.
  • a fusion protein contemplated herein comprises one or more DNA binding domains and one or more nucleases, and one or more linker and/or self-cleaving polypeptides.
  • a fusion protein contemplated herein comprises nuclease variant; a linker or self-cleaving peptide; and an end-processing enzyme including but not limited to a 5'- 3 ' exonuclease, a 5 ' -3 ' alkaline exonuclease, and a 3 ' -5 ' exonuclease ⁇ e.g., Trex2).
  • Fusion polypeptides can comprise one or more polypeptide domains or segments including, but are not limited to signal peptides, cell permeable peptide domains (CPP), DNA binding domains, nuclease domains, etc., epitope tags ⁇ e.g., maltose binding protein ("MBP"), glutathione S transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptide linkers, and polypeptide cleavage signals. Fusion polypeptides are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N- terminus, or N-terminus to C-terminus.
  • signal peptides e domains or segments
  • CPP cell permeable peptide domains
  • MBP maltose binding protein
  • GST glutathione S transferase
  • HIS6 HIS6, MYC
  • FLAG glutathione S
  • the polypeptides of the fusion protein can be in any order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be produced by chemical synthetic methods or by chemical linkage between the two moieties or may generally be prepared using other standard techniques.
  • Ligated DNA sequences comprising the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as disclosed elsewhere herein.
  • Fusion polypeptides may optionally comprises a linker that can be used to link the one or more polypeptides or domains within a polypeptide.
  • a peptide linker sequence may be employed to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structures so as to allow the polypeptide domains to exert their desired functions.
  • Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques in the art.
  • Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
  • Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence.
  • Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al. , Gene 40:39-46, 1985; Murphy et al, Proc. Natl. Acad. Sci.
  • Linker sequences are not required when a particular fusion polypeptide segment contains non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • Preferred linkers are typically flexible amino acid subsequences which are synthesized as part of a recombinant fusion protein.
  • Linker polypeptides can be between 1 and 200 amino acids in length, between 1 and 100 amino acids in length, or between 1 and 50 amino acids in length, including all integer values in between.
  • EGKSSGSGSESKVD (SEQ ID NO: 28) (Chaudhary et al. , 1990, Proc. Natl. Acad. Sci. U.S.A. 87: 1066-1070); KESGS VS SEQL AQFRSLD (SEQ ID NO: 29) (Bird et al, 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 30); LRQRDGERP (SEQ ID NO: 31);
  • LRQKDGGGSERP SEQ ID NO: 32
  • LRQKD(GGGS) 2 ERP SEQ ID NO: 33
  • flexible linkers can be rationally designed using a computer program capable of modeling both DNA-binding sites and the peptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91 : 11099-11103 (1994) or by phage display methods.
  • Fusion polypeptides may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein or between an endogenous open reading frame and a polypeptide encoded by a donor repair template.
  • a polypeptide cleavage site can be put into any linker peptide sequence.
  • Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).
  • Suitable protease cleavages sites and self-cleaving peptides are known to the skilled person (see, e.g., in Ryan et al, 1997. J Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594).
  • Exemplary protease cleavage sites include, but are not limited to the cleavage sites of poty virus NIa proteases (e.g., tobacco etch virus protease), poty virus HC proteases, poty virus PI (P35) proteases, byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus L proteases, enterovirus 2A proteases, rhinovirus 2A proteases, picorna 3C proteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungro spherical vims) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase.
  • poty virus NIa proteases e.g., tobacco etch virus protease
  • poty virus HC proteases pot
  • TEV tobacco etch virus protease cleavage sites
  • EXXYXQ(G/S) SEQ ID NO: 34
  • ENLYFQG SEQ ID NO: 35
  • ENLYFQS SEQ ID NO: 36
  • the self-cleaving polypeptide site comprises a 2A or 2A-like site, sequence or domain (Donnelly et al., 2001. J. Gen. Virol. 82: 1027-1041).
  • the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.
  • the viral 2A peptide is selected from the group consisting of: a foot-and-mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a Thosea asigna virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.
  • FMDV foot-and-mouth disease virus
  • EAV equine rhinitis A virus
  • TaV Thosea asigna virus
  • PTV-1 porcine teschovirus-1
  • Exemplary 2A sites include the following sequences:
  • polynucleotide or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA/RNA hybrids. Polynucleotides may be single-stranded or double-stranded and either recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA
  • RNA RNA
  • miRNA microRNA
  • ribozymes RNA
  • gRNA genomic RNA
  • RNA(+) minus strand RNA
  • RNA(-) minus strand RNA
  • crRNA crRNA
  • single guide RNA sgRNA
  • synthetic RNA synthetic mRNA
  • genomic DNA gDNA
  • PCR amplified DNA PCR amplified DNA
  • Polynucleotides refer to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, as well as all intermediate lengths.
  • intermediate lengths means any length between the quoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc. ; 151, 152, 153, etc.; 201, 202, 203, etc.
  • polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.
  • polynucleotides may be codon-optimized.
  • codon-optimized refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and/or activity of the polypeptide.
  • Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, (x) systematic variation of codon sets for each amino acid, and/or (xi) isolated removal of spurious translation initiation sites.
  • nucleotide refers to a heterocyclic nitrogenous base in N- glycosidic linkage with a phosphorylated sugar. Nucleotides are understood to include natural bases, and a wide variety of art-recognized modified bases. Such bases are generally located at the 1 ' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. In ribonucleic acid (RNA), the sugar is a ribose, and in deoxyribonucleic acid (DNA) the sugar is a deoxyribose, i.e., a sugar lacking a hydroxyl group that is present in ribose.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • Exemplary natural nitrogenous bases include the purines, adenosine (A) and guanidine (G), and the pyrimidines, cytidine (C) and thymidine (T) (or in the context of RNA, uracil (U)).
  • the C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine.
  • Nucleotides are usually mono, di- or triphosphates.
  • the nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, nucleotide derivatives, modified nucleotides, non-natural nucleotides, and non-standard nucleotides; see for example, WO 92/07065 and WO 93/15187).
  • modified nucleic acid bases are summarized by Limbach et al, (1994, Nucleic Acids Res. 22, 2183- 2196).
  • a nucleotide may also be regarded as a phosphate ester of a nucleoside, with esterification occurring on the hydroxyl group attached to C-5 of the sugar.
  • nucleoside refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases, and also to include well known modified bases. Such bases are generally located at the 1 ' position of a nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group.
  • the nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides, or non-standard nucleosides).
  • modified nucleic acid bases are summarized by Limbach et al., (1994, Nucleic Acids Res. 22, 2183-2196).
  • polynucleotides include, but are not limited to polynucleotides encoding SEQ ID NOs: 1-12 and 21 and polynucleotide sequences set forth in SEQ ID NOs:
  • polynucleotides contemplated herein include, but are not limited to polynucleotides encoding homing endonuclease variants, megaTALs, end- processing enzymes, fusion polypeptides, and expression vectors, viral vectors, and transfer plasmids comprising polynucleotides contemplated herein.
  • polynucleotide variant and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or modified, or replaced with different nucleotides.
  • a polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions.
  • stringent conditions describes hybridization protocols in which nucleotide sequences at least 60% identical to each other remain hybridized.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • sequence identity or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by- nucleotide basis or an amino acid-by -amino acid basis over a window of comparison.
  • a "percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Tip, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Tip, Lys, Arg,
  • nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.
  • references to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length.
  • two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • BESTFIT Pearson FASTA
  • FASTA Altschul et al.
  • TFASTA Pearson's Alignment of Altschul et al.
  • a detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al, Current Protocols in
  • isolated polynucleotide refers to a polynucleotide that has been purified from the sequences which flank it in a naturally -occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment.
  • isolated polynucleotide refers to a complementary DNA
  • cDNA a recombinant polynucleotide, a synthetic polynucleotide, or other polynucleotide that does not exist in nature and that has been made by the hand of man.
  • a polynucleotide comprises an mRNA encoding a polypeptide contemplated herein including, but not limited to, a homing endonuclease variant, a megaTAL, and an end-processing enzyme.
  • the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.
  • a mRNA contemplated herein comprises a 5 ' cap comprising a 5 ' -ppp-5 ' -triphosphate linkage between a terminal guanosine cap residue and the 5 ' -terminal transcribed sense nucleotide of the mRNA molecule. This 5 ' -guanylate cap may then be methylated to generate an N7-methyl-guanylate residue.
  • 5 ' cap suitable for use in particular embodiments of the mRNA polynucleotides contemplated herein include, but are not limited to: unmethylated 5 ' cap analogs, e.g., G(5 ' )ppp(5 ' )G, G(5 ' )ppp(5 ' )C, G(5 ' )ppp(5 ' )A; methylated 5 ' cap analogs, e.g., m 7 G(5 ' )ppp(5 ' )G, m 7 G(5 ' )ppp(5 ' )C, and m 7 G(5 ' )ppp(5 ' )A; dimethylated 5 ' cap analogs, e.g., m 2 ' 7 G(5 ' )ppp(5 ' )G, m 2,7 G(5 ' )ppp(5 ' )C, and m 2,7 G(5 ' cap analogs,
  • mRNAs comprise a 5 ' cap that is a 7-methyl guanylate ("m 7 G") linked via a triphosphate bridge to the 5 ' -end of the first transcribed nucleotide, resulting in m 7 G(5 ') ⁇ (5 ')N, where N is any nucleoside.
  • m 7 G 7-methyl guanylate
  • mRNAs comprise a 5 ' cap wherein the cap is a CapO structure (CapO structures lack a 2 ' -0-methyl residue of the ribose attached to bases 1 and 2), a Capl structure (Capl structures have a 2 ' -0-methyl residue at base 2), or a Cap2 structure (Cap2 structures have a 2 ' -0-methyl residue attached to both bases 2 and 3).
  • the cap is a CapO structure
  • CapO structures lack a 2 ' -0-methyl residue of the ribose attached to bases 1 and 2
  • a Capl structure Capl structures have a 2 ' -0-methyl residue at base 2
  • a Cap2 structure Cap2 structures have a 2 ' -0-methyl residue attached to both bases 2 and 3).
  • an mRNA comprises a m 7 G(5 )ppp(5 ')G cap.
  • an mRNA comprises an ARCA cap.
  • an mRNA contemplated herein comprises one or more modified nucleosides.
  • an mRNA comprises one or more modified nucleosides selected from the group consisting of: pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2- thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl -uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl- uridine, 1 -methyl -pseudouri dine, 4-thio- 1 -methyl -pseudouri dine, 2-thio-l-methyl
  • an mRNA comprises one or more modified nucleosides selected from the group consisting of: pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2- thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl -uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl- uridine, 1 -methyl -pseudouri dine, 4-thio-l -methyl -pseudouri dine, 2-thio-l-methyl
  • an mRNA comprises one or more modified nucleosides selected from the group consisting of: 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4- acetylcytidine, 5-formyl cyti dine, N4-methylcyti dine, 5-hydroxymethylcytidine, 1 -methyl - pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l -methyl -pseudoisocytidine, 4-thio-l - methyl-l-deaza-pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocyti dine, zebularine, 5-aza-
  • an mRNA comprises one or more modified nucleosides selected from the group consisting of: 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8- aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl
  • an mRNA comprises one or more modified nucleosides selected from the group consisting of: inosine, 1 -methyl -inosine, wyosine, wybutosine, 7-deaza- guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7- deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6- methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8- oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio- guanosine, and N2,N2-dimethyl-6-thio-guanosine.
  • an mRNA comprises one or more pseudouridines, one or more 5- methyl-cytosines, and/or one or more 5-methyl-cytidines.
  • an mRNA comprises one or more pseudouridines.
  • an mRNA comprises one or more 5-methyl-cytidines.
  • an mRNA comprises one or more 5-methyl-cytosines.
  • an mRNA contemplated herein comprises a poly(A) tail to help protect the mRNA from exonuclease degradation, stabilize the mRNA, and facilitate translation.
  • an mRNA comprises a 3' poly(A) tail structure.
  • the length of the poly(A) tail is at least about 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or at least about 500 or more adenine nucleotides or any intervening number of adenine nucleotides.
  • the length of the poly(A) tail is at least about 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 202, 203, 205, 206, 207, 208, 209,
  • the length of the poly(A) tail is about 10 to about 500 adenine nucleotides, about 50 to about 500 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 250 to about 500 adenine nucleotides, about 300 to about 500 adenine nucleotides, about 50 to about 450 adenine nucleotides, about 50 to about 400 adenine nucleotides, about 50 to about 350 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 100 to about 450 adenine nucleotides, about 100 to about 400 adenine nucleotides, about 100 to about 350 adenine nucleotides, about 100 to about 300 adenine nucleotides, about 150 to about 500 adenine nucleot
  • polynucleotide having a free hydroxyl (OH) group polynucleotide having a free hydroxyl (OH) group.
  • Polynucleotide sequences can be annotated in the 5 ' to 3 ' orientation or the 3 ' to 5 ' orientation.
  • the 5 ' to 3 ' strand is designated the “sense,” “plus,” or “coding” strand because its sequence is identical to the sequence of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA, instead of thymine (T) in DNA].
  • pre-mRNA pre-messenger
  • the complementary 3 ' to 5 ' strand which is the strand transcribed by the RNA polymerase is designated as "template,” “antisense,” “minus,” or “non-coding” strand.
  • the term “reverse orientation” refers to a 5' to 3' sequence written in the 3 ' to 5 ' orientation or a 3 ' to 5 ' sequence written
  • complementary and complementarity refer to polynucleotides ⁇ i.e., a sequence of nucleotides) related by the base-pairing rules.
  • the complementary strand of the DNA sequence 5' A G T C A T G 3' is 3' T C A G T A C 5'.
  • the latter sequence is often written as the reverse complement with the 5 ' end on the left and the 3 ' end on the right, 5 C A T G A C T 3 ' .
  • a sequence that is equal to its reverse complement is said to be a palindromic sequence.
  • Complementarity can be "partial,” in which only some of the nucleic acids ' bases are matched according to the base pairing rules.
  • nucleic acid cassette or “expression cassette” as used herein refers to genetic sequences within the vector which can express an RNA, and subsequently a polypeptide.
  • the nucleic acid cassette contains a gene(s)-of-interest, e.g., a polynucleotide(s)-of-interest.
  • the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, enhancer, poly(A) sequence, and a gene(s)-of-interest, e.g., a polynucleotide(s)-of-interest.
  • Vectors may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleic acid cassettes.
  • the nucleic acid cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or a polypeptide, undergo appropriate post-translational modifications required for activity in the transformed cell, and be translocated to the appropriate compartment for biological activity by targeting to appropriate intracellular compartments or secretion into extracellular compartments.
  • the cassette has its 3 ' and 5 ' ends adapted for ready insertion into a vector, e.g., it has restriction endonuclease sites at each end.
  • the nucleic acid cassette contains the sequence of a therapeutic gene used to treat, prevent, or ameliorate a genetic disorder.
  • the cassette can be removed and inserted into a plasmid or viral vector as a single unit.
  • Polynucleotides include polynucleotide(s)-of-interest.
  • polynucleotide-of-interest refers to a polynucleotide encoding a polypeptide or fusion polypeptide or a polynucleotide that serves as a template for the transcription of an inhibitory polynucleotide, as contemplated herein.
  • nucleotide sequences that may encode a polypeptide, or fragment of variant thereof, as contemplated herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene.
  • polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, for example polynucleotides that are optimized for human and/or primate codon selection.
  • polynucleotides comprising particular allelic sequences are provided. Alleles are endogenous polynucleotide sequences that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides.
  • a polynucleotide-of-interest comprises a donor repair template.
  • a polynucleotide-of-interest comprises an inhibitory polynucleotide including, but not limited to, an siRNA, an miRNA, an shRNA, a ribozyme or another inhibitory RNA.
  • a donor repair template comprising an inhibitory RNA comprises one or more regulatory sequences, such as, for example, a strong constitutive pol ⁇ , e.g., human or mouse U6 snRNA promoter, the human and mouse HI RNA promoter, or the human tRNA-val promoter, or a strong constitutive pol ⁇ promoter, as described elsewhere herein.
  • a strong constitutive pol ⁇ e.g., human or mouse U6 snRNA promoter, the human and mouse HI RNA promoter, or the human tRNA-val promoter, or a strong constitutive pol ⁇ promoter, as described elsewhere herein.
  • polynucleotides contemplated in particular embodiments may be combined with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Art sites), termination codons,
  • promoters and/or enhancers such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Art sites), termination codons,
  • transcriptional termination signals post-transcription response elements, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably. It is therefore contemplated in particular embodiments that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol .
  • Polynucleotides can be prepared, manipulated, expressed and/or delivered using any of a variety of well-established techniques known and available in the art.
  • a nucleotide sequence encoding the polypeptide can be inserted into appropriate vector.
  • a desired polypeptide can also be expressed by delivering an mRNA encoding the polypeptide into the cell.
  • vectors include, but are not limited to plasmid, autonomously replicating sequences, and transposable elements, e.g., Sleeping Beauty, PiggyBac.
  • vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses.
  • artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC)
  • bacteriophages such as lambda phage or Ml 3 phage
  • animal viruses include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses.
  • viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).
  • retrovirus including lentivirus
  • adenovirus e.g., adeno-associated virus
  • herpesvirus e.g., herpes simplex virus
  • poxvirus baculovirus
  • papillomavirus papillomavirus
  • papovavirus e.g., SV40
  • expression vectors include, but are not limited to pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DESTTM, pLenti6/V5- DESTTM, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
  • coding sequences of polypeptides disclosed herein can be ligated into such expression vectors for the expression of the polypeptides in mammalian cells.
  • the vector is an episomal vector or a vector that is maintained extrachromosomally.
  • episomal vector refers to a vector that is able to replicate without integration into host ' s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally.
  • “Expression control sequences,” “control elements,” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector— origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, post-transcriptional regulatory elements, a
  • polyadenylation sequence 5 ' and 3 ' untranslated regions—which interact with host cellular proteins to carry out transcription and translation.
  • Such elements may vary in their strength and specificity.
  • any number of suitable transcription and translation elements including ubiquitous promoters and inducible promoters may be used.
  • a polynucleotide comprises a vector, including but not limited to expression vectors and viral vectors.
  • a vector may comprise one or more exogenous, endogenous, or heterologous control sequences such as promoters and/or enhancers.
  • An "endogenous control sequence” is one which is naturally linked with a given gene in the genome.
  • An “exogenous control sequence” is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter.
  • a “heterologous control sequence” is an exogenous sequence that is from a different species than the cell being genetically manipulated.
  • a “synthetic" control sequence may comprise elements of one more endogenous and/or exogenous sequences, and/or sequences determined in vitro or in silico that provide optimal promoter and/or enhancer activity for the particular therapy.
  • promoter refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds.
  • An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter.
  • promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.
  • enhancer refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence.
  • An enhancer can function cooperatively or additively with promoters and/or other enhancer elements.
  • promoter/enhancer refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and/or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide- of-interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • constitutive expression control sequence refers to a promoter, enhancer, or promoter/enhancer that continually or continuously allows for transcription of an operably linked sequence.
  • a constitutive expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter/enhancer that allows expression in a wide variety of cell and tissue types or a "cell specific,” “cell type specific,” “cell lineage specific,” or “tissue specific” promoter, enhancer, or promoter/enhancer that allows expression in a restricted variety of cell and tissue types, respectively.
  • Illustrative ubiquitous expression control sequences suitable for use in particular embodiments include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and PI 1 promoters from vaccinia virus, a short elongation factor 1 -alpha (EF la-short) promoter, a long elongation factor 1 -alpha (EF la-long) promoter, early growth response 1 (EGRl), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1
  • a cell, cell type, cell lineage or tissue specific expression control sequence may be desirable to use to achieve cell type specific, lineage specific, or tissue specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues or during specific stages of development).
  • condition expression may refer to any type of conditional expression including, but not limited to, inducible expression; repressible expression;
  • conditional expression of a polynucleotide-of-interest e.g., expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that causes an increase or decrease in expression of the polynucleotide encoded by the polynucleotide-of-interest.
  • inducible promoters/sy stems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the "GeneSwitch” mifepristone-regulatable system (Sirin etal., 2003, Gene, 323:67), the cumate inducible gene switch (WO 2002/088346), tetracycline-dependent regulatory systems, etc.
  • steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the "GeneSwitch”
  • polynucleotides comprises at least one (typically two) site(s) for recombination mediated by a site specific recombinase.
  • recombinase or "site specific recombinase” include excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, six, seven, eight, nine, ten or more.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof.
  • Illustrative examples of recombinases suitable for use in particular embodiments include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, OC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCEl, and ParA.
  • the polynucleotides may comprise one or more recombination sites for any of a wide variety of site specific recombinases. It is to be understood that the target site for a site specific recombinase is in addition to any site(s) required for integration of a vector, e.g., a retroviral vector or lentiviral vector. As used herein, the terms "recombination sequence,"
  • recombination site or “site specific recombination site” refer to a particular nucleic acid sequence to which a recombinase recognizes and binds.
  • loxP which is a 34 base pair sequence comprising two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence (see FIG. 1 of Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)).
  • Other exemplary loxP sites include, but are not limited to: lox511 (Hoess et al.
  • Suitable recognition sites for the FLP recombinase include, but are not limited to: FRT (McLeod, et al, 1996), Fi,F 2 , F3 (Schlake and Bode, 1994), F 4 ,F 5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al, 1988), FRT(RE) (Senecoff et al, 1988).
  • recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme ⁇ Integrase, e.g., phi-c31.
  • the ⁇ C31 SSR mediates recombination only between the heterotypic sites attB (34 bp in length) and attP (39 bp in length) (Groth et al, 2000).
  • attB and attP named for the attachment sites for the phage integrase on the bacterial and phage genomes, respectively, both contain imperfect inverted repeats that are likely bound by ⁇ C31 homodimers (Groth et al, 2000).
  • the product sites, attL and attR, are effectively inert to further ⁇ > 1-mediated recombination (Belteki et al, 2003), making the reaction irreversible.
  • attB-bearing DNA inserts into a genomic attP site more readily than an attP site into a genomic attB site (Thyagaraj an et al. , 2001 ; Belteki et al. , 2003).
  • typical strategies position by homologous recombination an attP-bearing "docking site" into a defined locus, which is then partnered with an attB-bearing incoming sequence for insertion.
  • a polynucleotide contemplated herein comprises a donor repair template polynucleotide flanked by a pair of recombinase recognition sites.
  • the repair template polynucleotide is flanked by LoxP sites, FRT sites, or art sites.
  • polynucleotides contemplated herein include one or more polynucleotides-of-interest that encode one or more polypeptides.
  • the polynucleotide sequences can be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides.
  • an "internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene. See, e.g., Jackson et al, 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES generally employed by those of skill in the art include those described in U.S. Pat. No. 6,692,736.
  • IRES immunoglobulin heavy-chain binding protein
  • VEGF vascular endothelial growth factor
  • FGF-2 fibroblast growth factor 2
  • IGFII insulinlike growth factor
  • EMCV encephelomycarditis virus
  • IRES VEGF IRES
  • Picornaviridae Dicistroviridae and Flaviviridae species
  • HCV Friend murine leukemia virus
  • MoMLV Moloney murine leukemia virus
  • the IRES used in polynucleotides contemplated herein is an EMCV IRES.
  • the polynucleotides comprise polynucleotides that have a consensus Kozak sequence and that encode a desired polypeptide.
  • Kozak sequence refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA to the small subunit of the ribosome and increases translation.
  • the consensus Kozak sequence is (GCC)RCCATGG [SEQ ID NO: 59], where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48).
  • vectors comprise a polyadenylation sequence 3 Of a polynucleotide encoding a polypeptide to be expressed.
  • polyA site denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II.
  • Polyadenylation sequences can promote mRNA stability by addition of a poly(A) tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly(A) tail are unstable and are rapidly degraded.
  • Illustrative examples of poly (A) signals that can be used in a vector includes an ideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA), a bovine growth hormone poly(A) sequence (BGHpA), a rabbit ⁇ -globin poly(A) sequence (rPgpA), or another suitable heterologous or endogenous poly(A) sequence known in the art.
  • a polynucleotide or cell harboring the polynucleotide utilizes a suicide gene, including an inducible suicide gene to reduce the risk of direct toxicity and/or uncontrolled proliferation.
  • the suicide gene is not immunogenic to the host harboring the polynucleotide or cell.
  • a certain example of a suicide gene that may be used is caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).
  • polynucleotides comprise gene segments that cause the genetically modified cells contemplated herein to be susceptible to negative selection in vivo.
  • Negative selection refers to an infused cell that can be eliminated as a result of a change in the in vivo condition of the individual.
  • the negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound.
  • Negative selection genes include, but are not limited to: the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyl transferase (APRT) gene, and bacterial cytosine deaminase.
  • HSV-I TK Herpes simplex virus type I thymidine kinase
  • HPRT hypoxanthine phosphribosyltransferase
  • APRT cellular adenine phosphoribosyl transferase
  • genetically modified cells comprise a polynucleotide further comprising a positive marker that enables the selection of cells of the negative selectable phenotype in vitro.
  • the positive selectable marker may be a gene, which upon being introduced into the host cell, expresses a dominant phenotype permitting positive selection of cells carrying the gene.
  • Genes of this type are known in the art, and include, but are not limited to hygromycin-B phosphotransferase gene (hph) which confers resistance to hygromycin B, the amino glycoside phosphotransferase gene (neo or aph) from Tn5 which codes for resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drug resistance (MDR) gene.
  • hph hygromycin-B phosphotransferase gene
  • DHFR dihydrofolate reductase
  • ADA adenosine deaminase gene
  • MDR multi-drug resistance
  • the positive selectable marker and the negative selectable element are linked such that loss of the negative selectable element necessarily also is accompanied by loss of the positive selectable marker.
  • the positive and negative selectable markers are fused so that loss of one obligatorily leads to loss of the other.
  • An example of a fused polynucleotide that yields as an expression product a polypeptide that confers both the desired positive and negative selection features described above is a hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene yields a polypeptide that confers hygromycin B resistance for positive selection in vitro, and ganciclovir sensitivity for negative selection in vivo. See also the publications of PCT US91/08442 and PCT/US94/05601, by S. D. Lupton, describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable markers with negative selectable markers.
  • Preferred positive selectable markers are derived from genes selected from the group consisting of hph, nco, and gpt
  • preferred negative selectable markers are derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt.
  • Exemplary bifunctional selectable fusion genes contemplated in particular embodiments include, but are not limited to genes wherein the positive selectable marker is derived from hph or neo, and the negative selectable marker is derived from cytosine deaminase or a TK gene or selectable marker.
  • polynucleotides encoding one or more nuclease variants, megaTALs, end-processing enzymes, or fusion polypeptides may be introduced into hematopoietic cells, e.g., T cells, by both non-viral and viral methods.
  • delivery of one or more polynucleotides encoding nucleases and/or donor repair templates may be provided by the same method or by different methods, and/or by the same vector or by different vectors.
  • vector is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule.
  • the transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule.
  • a vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA.
  • non-viral vectors are used to deliver one or more polynucleotides contemplated herein to a T cell.
  • Illustrative examples of non-viral vectors include, but are not limited to plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.
  • Illustrative methods of non-viral delivery of polynucleotides contemplated in particular embodiments include, but are not limited to: electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene gun, and heat-shock.
  • polynucleotide delivery systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc.
  • Lipofection reagents are sold commercially (e.g.,
  • Viral vectors comprising polynucleotides contemplated in particular embodiments 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., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsy, etc.) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient.
  • viral vectors comprising nuclease variants and/or donor repair templates are administered directly to an organism for transduction of cells in vivo.
  • naked DNA 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. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • embodiments contemplated herein include, but are not limited to adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.
  • AAV adeno-associated virus
  • retrovirus retrovirus
  • herpes simplex virus adenovirus
  • vaccinia virus vectors vaccinia virus vectors.
  • one or more polynucleotides encoding a nuclease variant and/or donor repair template are introduced into a hematopoietic cell, e.g., a T cell, by transducing the cell with a recombinant adeno-associated virus (rAAV), comprising the one or more polynucleotides.
  • a hematopoietic cell e.g., a T cell
  • rAAV recombinant adeno-associated virus
  • AAV is a small (-26 nm) replication-defective, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell.
  • Recombinant AAV rAAV
  • rAAV are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5 ' and 3 ' AAV inverted terminal repeats (ITRs).
  • the ITR sequences are about 145 bp in length.
  • the rAAV comprises ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.
  • a chimeric rAAV is used the ITR sequences are isolated from one AAV serotype and the capsid sequences are isolated from a different AAV serotype.
  • a rAAV with ITR sequences derived from AAV2 and capsid sequences derived from AAV6 is referred to as AAV2/AAV6.
  • the rAAV vector may comprise ITRs from AAV2, and capsid proteins from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.
  • the rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV6.
  • the rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV2.
  • engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest.
  • Construction of rAAV vectors, production, and purification thereof have been disclosed, e.g., in U.S. Patent Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated by reference herein, in its entirety.
  • one or more polynucleotides encoding a nuclease variant and/or donor repair template are introduced into a hematopoietic cell, by transducing the cell with a retrovirus, e.g., lentivirus, comprising the one or more polynucleotides.
  • a retrovirus e.g., lentivirus
  • retrovirus refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome.
  • retroviruses suitable for use in particular embodiments include, but are not limited to: Moloney murine leukemia virus (M- MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus
  • HaMuSV murine mammary tumor virus
  • MoMTV gibbon ape leukemia virus
  • GaLV gibbon ape leukemia virus
  • FLV feline leukemia virus
  • BSV Rous Sarcoma Virus
  • lentivirus refers to a group (or genus) of complex retroviruses.
  • Illustrative lentiviruses include, but are not limited to: HIV (human
  • HIV based vector backbones i.e., HIV cis-acting sequence elements
  • HIV cis-acting sequence elements HIV based vector backbones
  • a lentiviral vector contemplated herein comprises one or more LTRs, and one or more, or all, of the following accessory elements: a cPPT/FLAP, a Psi ( ⁇ ) packaging signal, an export element, poly (A) sequences, and may optionally comprise a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as discussed elsewhere herein.
  • lentiviral vectors contemplated herein may be integrative or non-integrating or integration defective lentivirus.
  • integration defective lentivirus or "IDLV” refers to a lentivirus having an integrase that lacks the capacity to integrate the viral genome into the genome of the host cells. Integration-incompetent viral vectors have been described in patent application WO 2006/010834, which is herein incorporated by reference in its entirety.
  • HIV-1 pol gene suitable to reduce integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253
  • the HIV-1 integrase deficient pol gene comprises a D64V, Dl 161,
  • Dl 16A, E152G, or E152A mutation D64V, Dl 161, and E152G mutations; or D64V, Dl 16A, and El 52 A mutations.
  • the HIV-1 integrase deficient pol gene comprises a D64V mutation.
  • LTR long terminal repeat
  • FLAP element refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, etal, 2000, Cell, 101 : 173.
  • a lentiviral vector contains a FLAP element with one or more mutations in the cPPT and/or CTS elements.
  • a lentiviral vector comprises either a cPPT or CTS element.
  • a lentiviral vector does not comprise a cPPT or CTS element.
  • RNA export element refers to a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human
  • HIV immunodeficiency virus
  • RRE immunodeficiency virus rev response element
  • HPRE hepatitis B virus post- transcriptional regulatory element
  • expression of heterologous sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors.
  • posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid at the protein, e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al. , 1999, J. Virol., 73 :2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al, Mol. Cell. Biol, 5:3864); and the like (Liu et al, 1995, Genes Dev., 9: 1766).
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • HPRE hepatitis B virus
  • Lentiviral vectors preferably contain several safety enhancements as a result of modifying the LTRs.
  • Self-inactivating (SIN) vectors refers to replication-defective vectors, e.g., in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication.
  • An additional safety enhancement is provided by replacing the U3 region of the 5 ' LTR with a heterologous promoter to drive transcription of the viral genome during production of viral particles.
  • heterologous promoters examples include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.
  • SV40 viral simian virus 40
  • CMV cytomegalovirus
  • MoMLV Moloney murine leukemia virus
  • RSV Rous sarcoma virus
  • HSV herpes simplex virus
  • lentiviral vectors are produced according to known methods. See e.g., Kutner et al, BMC Biotechnol. 2009;9: 10. doi: 10.1186/1472-6750-9-10; Kutner et a/. Nat. Protoc. 2009;4(4):495-505. doi: 10.1038/nprot.2009.22.
  • most or all of the viral vector backbone sequences are derived from a lentivirus, e.g., HIV-1.
  • a lentivirus e.g., HIV-1.
  • many different sources of retroviral and/or lentiviral sequences can be used, or combined and numerous substitutions and alterations in certain of the lentiviral sequences may be accommodated without impairing the ability of a transfer vector to perform the functions described herein.
  • lentiviral vectors are known in the art, see Naldini et al, (1996a, 1996b, and 1998); Zufferey et al, (1997); Dull et al, 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which may be adapted to produce a viral vector or transfer plasmid contemplated herein.
  • one or more polynucleotides encoding a nuclease variant and/or donor repair template are introduced into a hematopoietic cell by transducing the cell with an adenovirus comprising the one or more polynucleotides.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Most adenovirus vectors are engineered such that a transgene replaces the Ad El a, Elb, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity.
  • adenovirus vectors which are replication deficient, may utilize a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones & Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham & Prevec, 1991 ).
  • a unique helper cell line designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones & Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham & Prevec, 1991 ).
  • Adenovirus vectors have been used in eukary otic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992).
  • Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al, 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz & Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al, 1993).
  • An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al, Hum. Gene Ther. 7: 1083-9 (1998)).
  • one or more polynucleotides encoding nuclease variant and/or donor repair template are introduced into a hematopoietic cell by transducing the cell with a herpes simplex virus, e.g., HSV-1, HSV-2, comprising the one or more polynucleotides.
  • a herpes simplex virus e.g., HSV-1, HSV-2
  • the mature HSV virion consists of an enveloped icosahedral capsid with a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb.
  • the HSV based viral vector is deficient in one or more essential or non-essential HSV genes.
  • the HSV based viral vector is replication deficient. Most replication deficient HSV vectors contain a deletion to remove one or more intermediate-early, early, or late HSV genes to prevent replication.
  • the HSV vector may be deficient in an immediate early gene selected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and a combination thereof.
  • HSV vectors are its ability to enter a latent stage that can result in long-term DNA expression and its large viral DNA genome that can accommodate exogenous DNA inserts of up to 25 kb.
  • HSV-based vectors are described in, for example, U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583, each of which are incorporated by reference herein in its entirety.
  • the genome edited cells manufactured by the methods contemplated in particular embodiments comprise one or more gene edits in an IL-lORa gene and provide improved cell- based therapeutics for the prevention, treatment, or amelioration of at least one symptom, of a cancer, GVHD, infectious disease, autoimmune disease, immunodeficiency or condition associated therewith.
  • a cancer GVHD
  • infectious disease infectious disease
  • autoimmune disease immunodeficiency or condition associated therewith.
  • compositions and methods contemplated herein increase the efficacy of adoptive cell therapies, in part, by making the therapeutic cells more resistant to immunosuppressive signals and exhaustion. It is also believed that the compositions and methods contemplated herein restore the potential of immune cells to respond to inflammatory and autoimmune diseases.
  • autologous/autogeneic self or non-autologous
  • non-self e.g., allogeneic, syngeneic or xenogeneic
  • autologous refers to cells from the same subject.
  • Allogeneic refers to cells of the same species that differ genetically to the cell in comparison.
  • Syngeneic refers to cells of a different subject that are genetically identical to the cell in comparison.
  • Xenogeneic refers to cells of a different species to the cell in comparison.
  • the cells are obtained from a mammalian subject.
  • the cells are obtained from a primate subject, optionally a non-human primate.
  • the cells are obtained from a human subject.
  • isolated cell refers to a non-naturally occurring cell, e.g., a cell that does not exist in nature, a modified cell, an engineered cell, etc., that has been obtained from an in vivo tissue or organ and is substantially free of extracellular matrix.
  • the term "population of cells” refers to a plurality of cells that may be made up of any number and/or combination of homogenous or heterogeneous cell types, as described elsewhere herein.
  • a population of cells may be isolated or obtained from peripheral blood.
  • a population of cells may comprise about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%), or about 100%> of the target cell type to be edited.
  • T cells may be isolated or purified from a population of heterogeneous cells using methods known in the art.
  • Illustrative examples of cell types whose genome can be edited using the compositions and methods contemplated herein include, but are not limited to, cell lines, primary cells, stem cells, progenitor cells, and differentiated cells, and mixtures thereof.
  • the genome editing compositions and methods are used to edit hematopoietic cells, more preferably immune cells, and even more preferably T cells.
  • T cell or "T lymphocyte” are art-recognized and are intended to include thymocytes, immune effector cells, regulatory T cells, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • a T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell.
  • the T cell can be a helper T cell (HTL; CD4 + T cell) CD4 + T cell, a cytotoxic T cell (CTL; CD8 + T cell), a tumor infiltrating cytotoxic T cell (TIL; CD8 + T cell), CD4 + CD8 + T cell, CD4 CD8 " T cell, or any other subset of T cells.
  • the T cell is an immune effector cell.
  • the T cell is a Treg.
  • the T cell is an NKT cell.
  • Other illustrative populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells.
  • genome edited cells comprise immune effector cells comprising an IL-lORa gene edited by the compositions and methods contemplated herein.
  • An "immune effector cell,” is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • Illustrative immune effector cells contemplated in particular embodiments are T lymphocytes, in particular cytotoxic T cells (CTLs; CD8 + T cells), TILs, and helper T cells (HTLs; CD4 + T cells).
  • immune effector cells include natural killer (NK) cells.
  • immune effector cells include natural killer T (NKT) cells.
  • T cells also include “regulatory T cells” or “Tregs.”
  • the terms “regulatory T cells” or “Tregs” are used interchangeably and refer to subsets of T cells that suppress immune and inflammatory responses to both self and foreign antigens.
  • Tregs suppress the proliferation or cytokine production of activated T cells.
  • Tregs directly suppress autoantibody production of autoreactive B cells.
  • Tregs modulate an inflammatory response by regulating activation of myeloid and endothelial cells.
  • Regulatory T cells are derived from the thymus (tTreg) or periphery (pTreg).
  • Tregs may be derived from CD4+ cells (CD4+ Tregs) or CD8+ cells (CD8+ Tregs).
  • Tregs express FoxP3 and cell surface markers including, but not limited, to CD4, CD25, GITR or CTLA4.
  • pTreg and tTreg subsets can also be identified on the basis of Helios expression.
  • Some regulatory T cell subsets, such as Trl cells, are FoxP3- deficient, and can be identified on the basis of CD49b and Lag3 expression.
  • Tregs can mediate immunosuppressive activity through both contact dependent (e.g., Granzyme B) or contact independent processes (e.g., by producing immunosuppressive cytokines, including but not limited to, IL10, IL35 and TGFb l).
  • contact dependent e.g., Granzyme B
  • contact independent processes e.g., by producing immunosuppressive cytokines, including but not limited to, IL10, IL35 and TGFb l.
  • Protent T cells and “young T cells,” are used interchangeably in particular embodiments and refer to T cell phenotypes wherein the T cell is capable of proliferation and a concomitant decrease in differentiation.
  • the young T cell has the phenotype of a "naive T cell.”
  • young T cells comprise one or more of, or all of the following biological markers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, and CD38.
  • young T cells comprise one or more of, or all of the following biological markers: CD62L, CD127, CD197, and CD38.
  • the young T cells lack expression of CD57, CD244, CD160, PD-1, CTLA4, ⁇ 3, and LAG3.
  • T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • a population of cells comprising immune effector cells or T cells comprises an edited IL-lORa gene, wherein the edit is a DSB repaired by HEJ.
  • an immune effector cell or T cell comprises an edited IL-lORa gene, wherein the edit is a DSB repaired by NHEJ.
  • the edit may be in a coding sequence of the IL- 10Ra gene, preferably in exon 2 of the IL-lORa gene, and more preferably at SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of the IL-lORa gene.
  • the edit is an insertion or deletion (INDEL) of 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 or more nucleotides in a coding sequence of the IL-lORa gene, preferably in exon 2 of the IL-lORa gene, more preferably at SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of the IL-lORa gene.
  • INDEL insertion or deletion
  • the edit is a deletion of 1, 2, 3, or 4 nucleotides in the coding sequence of the IL-lORa gene, preferably in exon 2 of the IL- lORa gene, more preferably at SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of the IL-lORa gene.
  • the edit is a deletion of about 1, 2, 3, or 4 nucleotides in a coding sequence of the IL-lORa gene, preferably in exon 2 of the IL-lORa gene, more preferably at SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of the IL-lORa gene.
  • SEQ ID NO: 13 or SEQ ID NO: 15
  • the edit is a deletion of 1, 2, 3, or 4 nucleotides in the coding sequence of the IL- lORa gene, preferably in exon 2 of the IL-lORa gene, more preferably at SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of the IL-lORa gene.
  • a population of cells comprising immune effector cells or T cells comprises an edited IL-lORa gene comprising a donor repair template incorporated at a DSB repaired by HDR.
  • the donor repair template may encode a FoxP3 polypeptide or polypeptide that increases or stabilizes FoxP3 expression, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
  • a population of cells comprising immune effector cells or T cells comprises an edited IL-lORa gene comprising a donor repair template comprising an IL- lORa gene or portion thereof and is designed to introduce one or more mutations in a genomic IL-lORa sequence such that a mutant IL-lORa gene product is expressed.
  • a population of cells comprising immune effector cells or T cells having one or more mutations in the IL-lORa gene that eliminate or substantial decrease IL-lORa expression is edited with a nuclease variant in the presence of a donor repair template designed to correct the one or more mutations and to increase or restore expression of IL-lORa.
  • T cells comprising one or more loss-of-function mutations, nonsense mutations, missense mutations, splice site mutations in the IL-lORa gene that eliminate or substantial decrease IL-lORa expression are edited with a nuclease variant in the presence of a donor repair template designed to correct the one or more mutations and to increase or restore expression of IL-lORa.
  • the donor template is designed such that a polynucleotide is inserted at a target site in the IL-lORa gene without substantially disrupting IL-lORa expression.
  • Illustrative examples of loss-of-function mutations in the IL-lORa gene that may be corrected by the genome edited compositions and methods contemplated here include, but are not limited to, W45G; Y64C; W69R; T84I; Y91C; V100G; R101W; Rl 17H; S138G; G141R; I169T; c.537G> A, p.T179T; g.IVS5+2T>C, c.690_765del, P206X; R262C, and E431X.
  • a genome edited cell comprises an edit in the IL-lORa gene and further comprises a polynucleotide encoding FoxP3, a bispecific T cell engager (BiTE) molecule; a cytokine (e.g., IL-2, insulin, IFN- ⁇ , IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-a), a chemokine (e.g., ⁇ - ⁇ , ⁇ - ⁇ , MCP-1, MCP-3, and RANTES), a cytotoxin (e.g., Perforin, Granzyme A, and Granzyme B), a cytokine receptor (e.g., an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, and an IL-21 receptor), or an engineered antigen receptor (e.g., an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a Daric receptor or components thereof
  • a donor repair template comprising the polynucleotide and a nuclease variant are introduced into the cell and the polynucleotide is incorporated into the cell' s genome at the DSB site in the IL-lORa gene by HDR repair.
  • the polynucleotide may also be introduced into the cell at a site other than the IL-lORa gene, e.g., by transducting the cell with a vector comprising the polynucleotide.
  • compositions contemplated in particular embodiments may comprise one or more polypeptides, polynucleotides, vectors comprising same, and genome editing compositions and genome edited cell compositions, as contemplated herein.
  • the genome editing compositions and methods contemplated in particular embodiments are useful for editing a target site in the human interleukin 10 receptor 1 alpha (IL-lORa) gene in a cell or a population of cells.
  • a genome editing composition is used to edit an IL-lORa gene in a hematopoietic cell, e.g., a T cell, an immune effector cell, or a Treg cell.
  • compositions contemplated herein comprise a nuclease variant, and optionally an end-processing enzyme, e.g., a 3 '-5' exonuclease (Trex2).
  • the nuclease variant may be in the form of an mRNA that is introduced into a cell via
  • compositions comprising an mRNA encoding a homing endonuclease variant or megaTAL, and optionally a 3 ' -5 ' exonuclease, is introduced in a cell via
  • the composition may be used to generate a genome edited cell or population of genome edited cells by error prone NHEJ.
  • the compositions contemplated herein comprise a donor repair template.
  • the composition may be delivered to a cell that expresses or will express nuclease variant, and optionally an end-processing enzyme.
  • the composition may be delivered to a cell that expresses or will express a homing endonuclease variant or megaTAL, and optionally a 3 '-5' exonuclease.
  • Expression of the gene editing enzymes in the presence of the donor repair template can be used to generate a genome edited cell or population of genome edited cells by HDR.
  • compositions contemplated herein comprise a population of cells, a nuclease variant, and optionally, a donor repair template.
  • compositions contemplated herein comprise a population of cells, a nuclease variant, an end-processing enzyme, and optionally, a donor repair template.
  • the nuclease variant and/or end-processing enzyme may be in the form of an mRNA that is introduced into the cell via polynucleotide delivery methods disclosed supra.
  • compositions contemplated herein comprise a population of cells, a homing endonuclease variant or megaTAL, and optionally, a donor repair template.
  • the compositions contemplated herein comprise a population of cells, a homing endonuclease variant or megaTAL, a 3 '-5' exonuclease, and optionally, a donor repair template.
  • the homing endonuclease variant, megaTAL, and/or 3 '-5 ' exonuclease may be in the form of an mRNA that is introduced into the cell via polynucleotide delivery methods disclosed supra.
  • the population of cells comprise genetically modified hematopoietic cells including, but not limited to, T cells, immune effector cells, and Tregs.
  • compositions include, but are not limited to pharmaceutical compositions.
  • composition refers to a composition formulated in pharmaceutically- acceptable or physiologically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions may be administered in combination with other agents as well, such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the composition.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic cells are administered.
  • pharmaceutical carriers can be sterile liquids, such as cell culture media, water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • composition comprising a pharmaceutically acceptable carrier is suitable for administration to a subject.
  • a pharmaceutically acceptable carrier is suitable for administration to a subject.
  • composition comprising a carrier is suitable for parenteral administration, e.g.,
  • a composition comprising a pharmaceutically acceptable carrier is suitable for intraventricular, intraspinal, or intrathecal administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media, or dispersions.
  • sterile aqueous solutions include sterile aqueous solutions, cell culture media, or dispersions.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is
  • compositions contemplated herein comprise genetically modified T cells and a pharmaceutically acceptable carrier.
  • a composition comprising a cell-based composition contemplated herein can be administered separately by enteral or parenteral administration methods or in combination with other suitable compounds to effect the desired treatment goals.
  • the pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the human subject being treated. It further should maintain or increase the stability of the composition.
  • the pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with other components of the composition.
  • the pharmaceutically acceptable carrier can be, without limitation, a binding agent ⁇ e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a filler ⁇ e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate, etc.), a lubricant ⁇ e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.), a disintegrant ⁇ e.g., starch, sodium starch glycolate, etc.), or a wetting agent ⁇ e.g., sodium lauryl sulfate, etc.).
  • a binding agent ⁇ e.g., pregelatinized
  • compositions contemplated herein include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins, hydroxymethylcelluloses, polyvinylpyrrolidones and the like.
  • buffers refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH.
  • buffers contemplated herein include, but are not limited to, Dulbecco ' s phosphate buffered saline (PBS), Ringer ' s solution, 5% dextrose in water (D5W), normal/physiologic saline (0.9% NaCl).
  • the pharmaceutically acceptable carriers may be present in amounts sufficient to maintain a pH of the composition of about 7.
  • the composition has a pH in a range from about 6.8 to about 7.4, e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4.
  • the composition has a pH of about 7.4.
  • compositions contemplated herein may comprise a nontoxic pharmaceutically acceptable medium.
  • the compositions may be a suspension.
  • the term "suspension” as used herein refers to non-adherent conditions in which cells are not attached to a solid support. For example, cells maintained as a suspension may be stirred or agitated and are not adhered to a support, such as a culture dish.
  • compositions contemplated herein are formulated in a suspension, where the genome edited T cells are dispersed within an acceptable liquid medium or solution, e.g., saline or serum-free medium, in an intravenous (IV) bag or the like.
  • acceptable liquid medium or solution e.g., saline or serum-free medium
  • IV intravenous
  • Acceptable diluents include, but are not limited to water, PlasmaLyte, Ringer ' s solution, isotonic sodium chloride (saline) solution, serum-free cell culture medium, and medium suitable for cryogenic storage, e.g., Cryostor® medium.
  • a pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin, and suitable for storing a composition comprising a population of genome edited T cells.
  • the therapeutic composition is intended to be administered into a human patient, and thus is substantially free of cell culture components such as bovine serum albumin, horse serum, and fetal bovine serum.
  • compositions are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects.
  • the pharmaceutically acceptable cell culture medium is a serum free medium.
  • Serum-free medium has several advantages over serum containing medium, including a simplified and better defined composition, a reduced degree of contaminants, elimination of a potential source of infectious agents, and lower cost.
  • the serum-free medium is animal-free, and may optionally be protein-free.
  • the medium may contain biopharmaceutically acceptable recombinant proteins.
  • Animal-free medium refers to medium wherein the components are derived from non-animal sources. Recombinant proteins replace native animal proteins in animal- free medium and the nutrients are obtained from synthetic, plant or microbial sources.
  • Protein-free in contrast, is defined as substantially free of protein.
  • serum-free media used in particular compositions includes, but is not limited to QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO lO.
  • compositions comprising genome edited T cells are formulated in PlasmaLyte.
  • compositions comprising genome edited T cells are formulated in a cryopreservation medium.
  • cryopreservation media with cryopreservation agents may be used to maintain a high cell viability outcome post-thaw.
  • cryopreservation media used in particular compositions includes, but is not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.
  • compositions are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS10.
  • the composition is substantially free of mycoplasma, endotoxin, and microbial contamination.
  • substantially free with respect to endotoxin is meant that there is less endotoxin per dose of cells than is allowed by the FDA for a biologic, which is a total endotoxin of 5 EU/kg body weight per day, which for an average 70 kg person is 350 EU per total dose of cells.
  • compositions comprising hematopoietic stem or progenitor cells transduced with a retroviral vector contemplated herein contain about 0.5 EU/mL to about 5.0 EU/mL, or about 0.5 EU/mL, 1.0 EU/mL, 1.5 EU/mL, 2.0 EU/mL, 2.5 EU/mL, 3.0 EU/mL, 3.5 EU/mL, 4.0 EU/mL, 4.5 EU/mL, or 5.0 EU/mL.
  • compositions and formulations suitable for the delivery of polynucleotides are contemplated including, but not limited to, one or more mRNAs encoding one or more reprogrammed nucleases, and optionally end-processing enzymes.
  • Exemplary formulations for ex vivo delivery may also include the use of various transfection agents known in the art, such as calcium phosphate, electroporation, heat shock and various liposome formulations (i.e., lipid-mediated transfection).
  • transfection agents such as calcium phosphate, electroporation, heat shock and various liposome formulations (i.e., lipid-mediated transfection).
  • Liposomes as described in greater detail below, are lipid bilayers entrapping a fraction of aqueous fluid. DNA spontaneously associates to the external surface of cationic liposomes (by virtue of its charge) and these liposomes will interact with the cell membrane.
  • formulation of pharmaceutically-acceptable carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., enteral and parenteral, e.g., intravascular, intravenous, intrarterial, intraosseously, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation.
  • enteral and parenteral e.g., intravascular, intravenous, intrarterial, intraosseously, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation.
  • enteral and parenteral e.g., intravascular, intravenous, intrarterial, intraosseously, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation.
  • particular embodiments contemplated herein
  • the genome edited cells manufactured by the methods contemplated in particular embodiments provide improved drug products for use in the prevention, treatment, or amelioration of at least one symptom of a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.
  • drug product refers to genetically modified cells produced using the compositions and methods contemplated herein.
  • the drug product comprises genetically modified immune effector cells or T cells.
  • an effective amount of genome edited immune effector cells or T cells comprising an edited IL-lORa gene are administered to a subject to prevent, treat, or ameliorate at least one symptom of a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.
  • the IL-lORa edited cells do not substantially express, or lack expression of, IL-lORa and therefore lack or substantially lack functional IL-lORa expression, e.g., lack the ability to increase T cell exhaustion and to inhibit expression of MHC class II molecules, costimulatory molecules, and proinflammatory cytokines.
  • genome edited immune effector cells that lack IL-lORa are more resistant to immunosuppressive signals from the tumor microenvironment.
  • a method of preventing, treating, or ameliorating at least one symptom of a cancer comprises administering the subject an effective amount of genome edited immune effector cells or T cells comprising an edited IL-lORa gene and an engineered TCR, CAR, or Daric, or other therapeutic transgene to redirect the cells to a tumor or cancer.
  • the genetically modified cells are a more durable and persistant drug product because the cells are more resistant to immunosuppressive signals from the tumor microenvironment by virtue of editing the IL-lORa gene to decrease or eliminate IL-lORa expression.
  • genome edited cells contemplated herein are used in the treatment of solid tumors or cancers.
  • genome edited cells contemplated herein are used in the treatment of solid tumors or cancers including, but not limited to: adrenal cancer,
  • adrenocortical carcinoma anal cancer, appendix cancer, astrocytoma, atypical
  • teratoid/rhabdoid tumor basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, bronchial tumors, cardiac tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer,
  • craniopharyngioma ductal carcinoma in situ (DCIS) endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous histiosarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), germ cell tumors, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular cancer, hypopharyngeal cancer, intraocular melanoma, kaposi sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid tumor, malignant mesot
  • nasopharyngeal cancer neuroblastoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell tumors, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, stomach cancer, sweat gland carcinoma, synovioma, testicular cancer, throat cancer, thymus cancer, thyroid cancer, urethral cancer, uterine
  • genome edited cells contemplated herein are used in the treatment of solid tumors or cancers including, without limitation, liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.
  • genome edited cells contemplated herein are used in the treatment of various cancers including but not limited to pancreatic, bladder, and lung.
  • genome edited cells contemplated herein are used in the treatment of liquid cancers or hematological cancers.
  • genome edited cells contemplated herein are used in the treatment of B-cell malignancies, including but not limited to: leukemias, lymphomas, and multiple myeloma.
  • genome edited cells contemplated herein are used in the treatment of liquid cancers including, but not limited to leukemias, lymphomas, and multiple myelomas: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic, promyelocyte, myelomonocytic, monocytic, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,
  • ALL acute
  • an effective amount of T cells comprising an IL-lORa gene edited using HDR to restore IL-lORa expression and/or increase or stabilize expression and/or function of FoxP3 or a polypeptide that enhances development, stability, and/or functionality of Treg cells is administered to a subject to prevent, treat, or ameliorate at least one symptom of GVHD, transplant rejection, an autoimmune disease or an inflammatory disease.
  • the genome edited cells are regulatory T cells (Tregs). Restoring IL-lORa expression in Treg cells would restore the cells' function of maintaining immune tolerance and immune system homeostasis. Enhancing FoxP3 function in Tregs is contemplated to enhance development, stability, and/or functionality of Treg cells.
  • Illustrative examples of diseases treated with genome edited Treg cells comprising an IL-lORa gene edited to restore IL-lORa expression and/or increase or stabilize expression and/or function of FoxP3 or a polypeptide that enhances development, stability, and/or functionality of Treg cells include, but are not limited to: Hashimoto' s thyroiditis, Grave' s disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, GVHD, transplant rejection, arthritis, and inflammatory bowel disease (IBD).
  • Hashimoto' s thyroiditis Grave' s disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus,
  • the GVHD is associated with solid organ transplants.
  • the solid organ transplant is selected from the group consisting of: a heart transplant, a lung transplant, a kidney transplant, a pancreas transplant, and a liver transplant.
  • the individual is administered the genome edited Treg cells contemplated herein to decrease GVHD while simultaneously maintaining or augmenting a GVL response post-transplant, e.g., bone marrow transplant.
  • Allogeneic lymphocytes produce a strong graft-versus-leukemia (GVL) effect, but the beneficial effect is limited by graft-versus- host disease (GVHD).
  • GVHD graft-versus- host disease
  • particular embodiments contemplate that administration of the genome edited Tregs will produce a GVL effect while suppressing GVHD.
  • Illustrative examples of IBD treated with genome edited Treg cells comprising an IL- lORa gene edited to restore IL-lORa expression and/or increase or stabilize expression and/or function of FoxP3 or a polypeptide that enhances development, stability, and/or functionality of Treg cells include, but are not limited to: ulcerative colitis, early onset ulcerative colitis, very early onset ulcerative colitis, pancolitis, Crohn's disease, and neonatal-onset Crohn's disease.
  • an effective amount of T cells comprising an IL-lORa gene edited to maintain or restore IL-lORa expression and introduce a polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells is administered to a subject to prevent, treat, or ameliorate at least one symptom of GVHD, an autoimmune disease, an inflammatory disease, or an immunodeficiency.
  • the polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells is inserted into the IL-lORa gene at DSB introduced by a nuclease variant and repaired by HDR
  • FoxP3 expression in particular T cells can induce and/or stabilize a Treg phenotype.
  • Illustrative examples of diseases treated with genome edited Treg cells comprising an IL-lORa gene edited to maintain or restore IL-lORa expression and introduce a polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells include, but are not limited to:
  • GVHD GVHD
  • transplant rejection arthritis
  • IBD inflammatory bowel disease
  • Illustrative examples of IBD treated with genome edited Treg cells comprising an IL- lORa gene edited to maintain or restore IL-lORa expression and introduce a polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells include, but are not limited to:
  • ulcerative colitis early onset ulcerative colitis, very early onset ulcerative colitis, pancolitis, Crohn's disease, and neonatal-onset Crohn's disease.
  • the Tregs are edited with a polynucleotide encoding an exogenous promoter operably linked to a polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
  • the Tregs are edited with a polynucleotide encoding a T2A or other viral self-cleaving peptide fused or linked to a polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
  • Preferred cells for use in the genome editing methods contemplated herein include autologous/autogeneic ("self) cells, preferably hematopoietic cells, more preferably T cells, and more preferably immune effector cells or Treg cells.
  • self autologous/autogeneic
  • methods comprising administering a therapeutically effective amount of genome edited cells contemplated herein or a composition comprising the same, to a patient in need thereof, alone or in combination with one or more therapeutic agents, are provided.
  • the cells are used in the treatment of patients at risk for developing a cancer, GVHD, transplant rejection, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.
  • particular embodiments comprise the treatment or prevention or amelioration of at least one symptom of a a cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency comprising administering to a subject in need thereof, a therapeutically effective amount of the genome edited cells contemplated herein.
  • a method of treating a cancer, GVHD, transplant rejection, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency in a subject in need thereof comprises administering an effective amount, e.g., therapeutically effective amount of a composition comprising genome edited cells contemplated herein.
  • an effective amount e.g., therapeutically effective amount of a composition comprising genome edited cells contemplated herein.
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
  • the effective amount of genome edited cells provided to a subject is at least 2 x 10 6 cells/kg, at least 3 x 10 6 cells/kg, at least 4 x 10 6 cells/kg, at least 5 x 10 6 cells/kg, at least 6 x 10 6 cells/kg, at least 7 x 10 6 cells/kg, at least 8 x 10 6 cells/kg, at least 9 x 10 6 cells/kg, or at least 10 x 10 6 cells/kg, or more cells/kg, including all intervening doses of cells.
  • the effective amount of genome edited cells provided to a subject is about 2 x 10 6 cells/kg, about 3 x 10 6 cells/kg, about 4 x 10 6 cells/kg, about 5 x 10 6 cells/kg, about 6 x 10 6 cells/kg, about 7 x 10 6 cells/kg, about 8 x 10 6 cells/kg, about 9 x 10 6 cells/kg, or about 10 x 10 6 cells/kg, or more cells/kg, including all intervening doses of cells.
  • the effective amount of genome edited cells provided to a subject is from about 2 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 3 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 4 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 5 x 10 6 cells/kg to about 10 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 7 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 8 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 7 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 8 x 10 6 cells/kg, 4 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 4 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 4 x 10 6 cells/kg
  • compositions contemplated in particular embodiments may be required to effect the desired therapy.
  • a composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more.
  • T cells can be activated from blood draws of from lOcc to 400cc.
  • T cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, lOOcc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more.
  • compositions contemplated in particular embodiments may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, compositions are administered parenterally.
  • parenteral administration and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.
  • a method of treating a subject diagnosed with a cancer comprises removing immune effector cells from the subject, editing the genome of said immune effector cells and producing a population of genome edited immune effector cells, and administering the population of genome edited immune effector cells to the same subject.
  • the immune effector cells comprise T cells.
  • the methods for administering the cell compositions contemplated in particular embodiments include any method which is effective to result in reintroduction of ex vivo genome edited immune effector cells or on reintroduction of the genome edited progenitors of immune effector cells that on introduction into a subject differentiate into mature immune effector cells.
  • One method comprises genome editing peripheral blood T cells ex vivo and returning the transduced cells into the subject.
  • I-Onul was reprogrammed to target exon 2 of the IL-lORa gene by constructing modular libraries containing variable amino acid residues in the DNA recognition interface.
  • degenerate codons were incorporated into I-Onul DNA binding domains using oligonucleotides.
  • the oligonucleotides encoding the degenerate codons were used as PCR templates to generate variant libraries by gap recombination in the yeast strain S. cerevisiae.
  • Each variant library spanned either the N- or C-terminal I-Onul DNA recognition domain and contained ⁇ 10 7 to 10 8 unique transformants.
  • the resulting surface display libraries were screened by flow cytometry for cleavage activity against target sites comprising the corresponding domains "half-sites" (SEQ ID NOs: 16-17).
  • Yeast displaying the N- and C-terminal domain reprogrammed I-Onul HEs were purified and the plasmid DNA was extracted. PCR reactions were performed to amplify the reprogrammed domains, which were subsequently transformed into S. cerevisiae to create a library of reprogrammed domain combinations. Fully reprogrammed I-Onul variants that recognize the complete target site (SEQ ID NO: 13) present in exon 2 of the IL-lORa gene were identified from this library and purified.
  • the activity of reprogrammed I-Onul HEs that target exon 2 of the IL-lORa gene was measured using a chromosomally integrated fluorescent reporter system (Certo et. al, 2011).
  • Fully reprogrammed I-Onul HEs that bind and cleave the IL-lORa target sequence were cloned into mammalian expression plasmids and then individually transfected into a HEK 293T fibroblast cell line that was reprogrammed to contain the IL-lORa target sequence upstream of an out-of-frame gene encoding the fluorescent mCherry protein.
  • a secondary I-Onul variant library was generated by performing random mutagenesis on one of the reprogrammed I-Onul HEs that targets the IL-lORa target site, identified in the initial reporter screen (IL-10Ra.G7, SEQ ID NO: 6). In addition, display-based flow sorting was performed under more stringent affinity conditions (50 pM) to isolate variants with improved binding characteristics.
  • Figure 3. This process identified an I-Onul variant, IL- lORa. G7. A3 (SEQ ID NO: 7), which has an approximately 2-fold higher rate of mCherry expressing cells than the parental I-Onul variant.
  • Figure 3 (middle panel). Random
  • I-Onul variant IL-lORa. G7. A3 under more stringent cleavage conditions (pH of 6.8) to isolate variants with improved cleavage activity.
  • This process identified an I-Onul variant, IL-10Ra.G7.A3.G7 (SEQ ID NO: 8), which has an approximately 33% higher rate of mCherry expressing cells than the parental I-Onul variant.
  • Figure 3 (lower panel).
  • IL-10Ra.G7.A3.G7 has subnanomolar affinity for the exon 2 target site (Figure 4).
  • Figure 5 shows the relative alignments of representative I-Onul variants as well as the positional information of the residues comprising the DNA recognition interface.
  • the I-Onul variant IL-10Ra.G7.A3.G7 was formatted as a megaTAL by appending an N-terminal 10.5 TAL array (SEQ ID NOs: 11 and 19) corresponding to an 11 base pair TAL array target site upstream of the IL-lORa LHE variant target site (SEQ ID NO: 14), using methods described in Boissel etal., 2013. Figure 6A.
  • Another version of the megaTAL comprises a C-terminal fusion to Trex2 via a linker sequence (SEQ ID NO: 12).
  • IL-10Ra.G7.A3.G7 megaTAL mRNA was prepared by in vitro transcription and co- transcriptionally capped with Anti-Reverse Cap Analog (ARC A) and enzymatically polyadenylated with poly(A) polymerase. The mRNA was purified and used to measure IL- 10Ra.G7.A3.G7 editing efficiency in primary human T cells.
  • ARC A Anti-Reverse Cap Analog
  • IL-10Ra.G7.A3.G7 megaTAL mRNA was prepared by in vitro transcription and co- transcriptionally capped with Anti-Reverse Cap Analog (ARC A) and enzymatically polyadenylated with poly(A) polymerase.
  • the mRNA was purified and used to measure IL- 10Ra.G7.A3.G7 editing efficiency in primary human T cells.
  • PBMC Primary human Peripheral blood mononuclear cells
  • CD3 and anti-CD28 antibodies were cultured in the presence of 250U/mL IL-2.
  • IL-10Ra.G7.A3.G7 megaTAL mRNA SEQ ID NO: 19
  • Trex2 exonuclease SEQ ID NO: 20.
  • Transfected T cells were expanded for additional 7-10 days and editing efficiency was measured using sequencing across the IL-lORa target site.
  • the frequency of small insertion/deletion (indel) events across the IL-lORa target site was measured using Tracking of Indels by DEcomposition (TIDE, see Brinkman et al., 2014).
  • Figure 6B shows a
  • Adeno-associated virus (AAV) plasmids containing transgene cassettes comprising a promoter, a transgene encoding a fluorescent protein, and a polyadenylation signal (SEQ ID NO: 22) were designed and constructed. The integrity of AAV ITR elements was confirmed with Xmal digest. The transgene cassette was placed between two 300bp homology regions flanking the ILlORa megaTAL cleavage site (SEQ ID NO: 15). Neither homology region contained the complete megaTAL target site.
  • Exemplary expression cassettes contain myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer- binding site substituted (MND) promoter operably linked to a polynucleotide encoding a fluorescent polypeptide, e.g., blue fluorescent protein (BFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), etc., and a WPRE
  • BFP blue fluorescent protein
  • RFP red fluorescent protein
  • CFP cyan fluorescent protein
  • GFP green fluorescent protein
  • FIG. 7A megaTAL-induced homologous recombination was evaluated in primary human T cells activated with CD3 and CD28 and cultured in complete media supplemented with JL-2. After 3 days, T cells were washed and electroporated with in vitro transcribed mRNA encoding the IL10Roc.G7.A3.G7 megaTAL (SEQ ID NO: 19), and subsequently transduced with purified recombinant AAV encoding MND-GFP transgene cassette (SEQ ID NO: 22). Flow cytometry was used at multiple time points to measure the frequency of T cells expressing the fluorescent protein and to differentiate transient expression of the fluorescent protein from the non-integrated rAAV targeting vector.

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Abstract

The invention provides improved genome editing compositions and methods for editing an IL-10Rα gene. The invention further provides genome edited cells for the prevention, treatment, or amelioration of at least one symptom of, a cancer, GVHD, a transplant rejection, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.

Description

IL-10 RECEPTOR ALPHA HOMING ENDONUCLEASE VARIANTS,
COMPOSITIONS, AND METHODS OF USE
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/411,154, filed October 21, 2016, and U.S. Provisional Application No. 62/375,751, filed August 16, 2016, each of which is incorporated by reference herein in its entirety.
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is BLBD_073_02WO_ST25.txt. The text file is 100 KB, was created on August 15, 2017, and is being submitted electronically via EFS-Web, concurrent with the filing of the specification.
BACKGROUND
Technical Field
The present invention relates to improved genome editing compositions. More particularly, the invention relates to nuclease variants, compositions, and methods of using same for editing the human interleukin 10 receptor 1 alpha (IL-lORa) gene.
Description of the Related Art Interleukin- 10 (IL-10) is an immunomodulatory pleiotropic cytokine produced by B cells, T cells, and cells of the monocyte/macrophage lineage and exhibits diverse activities on various cell types in the immune system. IL-10 signaling is mediated through an IL-IO/IL- lORa/β receptor signaling complex and is associated with immunosuppression, whereas lack of IL-10 signaling is associated with autoimmune disease.
IL-10 signaling is associated with immunosuppressive tumor microenvironments and cancers having a poor prognosis, e.g., melanoma, multiple myeloma. IL-10
immunosuppression may limit the magnitude of T cell responses by inhibiting expression of MHC class II molecules, costimulatory molecules, and proinflammatory cytokines, e.g., including, but not limited to, tumor necrosis factor-alpha (TNFa), interleukin-6 (IL-6) and interleukin-1 beta (IL-Ιβ), in antigen presenting cells (APCs).
IL-10 signaling also plays a role in immune cell function and homeostasis. Immune cells that express IL- 1 OR have the ability to suppress immune responses, including
inflammatory disease, autoimmune responses, etc. In contrast, immune cells that have a loss- of-function mutation in IL-lORa may more susceptible to immune disorders. For example, disruption of IL-lORa in gut macrophages is associated with susceptibility to autoimmune diseases. In addition, disruption of IL-lORa in regulatory T cells (Tregs) deregulates Treg function and leads to severe autoimmune colitis.
BRIEF SUMMARY
The invention generally relates, in part, to compositions comprising homing endonuclease variants and megaTALs that cleave a target site in the human IL-lORa gene and methods of using the same.
In various embodiments, the present invention contemplates, in part, a polypeptide comprising a homing endonuclease (HE) variant that cleaves a target site in the human interleukin 10 receptor 1 alpha (IL-lORa) gene.
In particular embodiments, the HE variant is an LAGLIDADG homing endonuclease (LHE) variant.
In certain embodiments, the polypeptide comprises a biologically active fragment of the HE variant.
In some embodiments, the biologically active fragment lacks the 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal amino acids compared to a corresponding wild type HE. In additional embodiments, the biologically active fragment lacks the 4 N-terminal amino acids compared to a corresponding wild type HE.
In certain embodiments, the biologically active fragment lacks the 8 N-terminal amino acids compared to a corresponding wild type HE.
In particular embodiments, the biologically active fragment lacks the 1, 2, 3, 4, or 5 C- terminal amino acids compared to a corresponding wild type HE.
In particular embodiments, wherein the biologically active fragment lacks the C- terminal amino acid compared to a corresponding wild type HE.
In some embodiments, the biologically active fragment lacks the 2 C-terminal amino acids compared to a corresponding wild type HE.
In further embodiments, the HE variant is a variant of an LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, I-ApaMI, I-CapHI, I-CapIV, I-CkaMI, I-CpaMI, I- CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-Gpil, I- GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I- Ncrl, I-NcrMI, I-OheMI, I-Onul, I-OsoMI, I-OsoMII, I-OsoMni, I-OsoMIV, I-PanMI, I- PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, and I-Vdil41I.
In particular embodiments, the HE variant is a variant of an LHE selected from the group consisting of: I-CpaMI, I-HjeMI, I-Onul, I-PanMI, and SmaMI.
In further embodiments, the HE variant is an I-Onul LHE variant.
In additional embodiments, the HE variant comprises one or more amino acid substitutions in the DNA recognition interface at amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
In particular embodiments, the HE variant comprises at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions in the DNA recognition interface at amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
In particular embodiments, the HE variant comprises at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions in at least one position selected from the position group consisting of positions: 24, 26, 28, 30, 32, 34, 36, 37, 38, 40, 41, 42, 44, 46, 48, 59, 70, 72, 75, 78, 80, 82, 138, 143, 145, 159, 168, 180, 182, 184, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 207, 223, 225, 227, 228, 229, 232, 236, 238, and 240 of any one of SEQ ID NOs: 1-5, or a biologically active fragment thereof.
In some embodiments, the HE variant comprises at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46V, Q46I, T48G, N59S, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180H, C180Y, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K, and V238I, in reference to an I- Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
In certain embodiments, the HE variant comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46V, T48G, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, S159P, F168L, C180H, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, K229A, F232K, D236K, and V238I, in reference to an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
In particular embodiments, the HE variant comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46I, T48G, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180H, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K, and V238I, in reference to an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
In further embodiments, the HE variant comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46I, T48G, N59S, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180Y, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K, and V238I, in reference to an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
In additional embodiments, the HE variant comprises an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 6-8, or a biologically active fragment thereof.
In particular embodiments, the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.
In particular embodiments, the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 7, or a biologically active fragment thereof.
In some embodiments, the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 8, or a biologically active fragment thereof.
In further embodiments, the polypeptide further comprises a DNA binding domain.
In some embodiments, the DNA binding domain is selected from the group consisting of: a TALE DNA binding domain and a zinc finger DNA binding domain.
In certain embodiments, the TALE DNA binding domain comprises about 9.5 TALE repeat units to about 15.5 TALE repeat units.
In additional embodiments, the TALE DNA binding domain binds a polynucleotide sequence in the IL-lORa gene.
In particular embodiments, the TALE DNA binding domain binds the polynucleotide sequence set forth in SEQ ID NO: 11.
In certain embodiments, the polypeptide binds and cleaves the polynucleotide sequence set forth in SEQ ID NO: 13. In certain embodiments, the zinc finger DNA binding domain comprises 2, 3, 4, 5, 6, 7, or 8 zinc finger motifs.
In further embodiments, the polypeptide further comprises a peptide linker and an end- processing enzyme or biologically active fragment thereof.
In particular embodiments, the polypeptide further comprises a viral self-cleaving 2A peptide and an end-processing enzyme or biologically active fragment thereof.
In additional embodiments, the end-processing enzyme or biologically active fragment thereof has 5 '-3 ' exonuclease, 5 '-3 ' alkaline exonuclease, 3 '-5' exonuclease, 5' flap endonuclease, helicase or template-independent DNA polymerase activity.
In particular embodiments, the end-processing enzyme comprises Trex2 or a biologically active fragment thereof.
In certain embodiments, the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-11, or a biologically active fragment thereof.
In further embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 9, or a biologically active fragment thereof.
In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 10, or a biologically active fragment thereof.
In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 11, or a biologically active fragment thereof.
In further embodiments, the polypeptide cleaves the human IL-lORa gene at the polynucleotide sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 15.
In various embodiments, the present invention contemplates, in part, a polynucleotide encoding a polypeptide contemplate herein.
In particular embodiments, the present invention contemplates, in part, an mRNA encoding a polypeptide contemplated herein.
In particular embodiments, the mRNA comprises the sequence set forth in SEQ ID NO: 19.
In various embodiments, the present invention contemplates, in part, a cDNA encoding a polypeptide contemplated herein. In certain embodiments, the present invention contemplates, in part, a vector comprising a polynucleotide encoding a polypeptide contemplated herein.
In various embodiments, the present invention contemplates, in part, a cell comprising a polypeptide contemplated herein.
In some embodiments, the present invention contemplates, in part, a cell comprising a polynucleotide encoding a polypeptide contemplated herein.
In various embodiments, the present invention contemplates, in part, a cell comprising a vector contemplated herein.
In additional embodiments, the present invention contemplates, in part, a cell comprising one or more genome modifications introduced by a polypeptide contemplated herein.
In some embodiments, the cell is a hematopoietic cell.
In additional embodiments, the cell is a T cell.
In particular embodiments, the cell is a CD3+, CD4+, and/or CD8+ cell.
In particular embodiments, the cell is an immune effector cell.
In further embodiments, the cell is a cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cells.
In certain embodiments, the cell is a natural killer (NK) cell or natural killer T ( KT) cell.
In additional embodiments, the cell is a regulatory T cell (Treg).
In particular embodiments, the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
In further embodiments, the cell comprises a polynucleotide encoding an engineered antigen receptor.
In particular embodiments, the engineered antigen receptor is selected from the group consisting of: an engineered T cell receptor, a chimeric antigen receptor, a DARIC, or a zetakine.
In certain embodiments, the cell comprises a polynucleotide encoding a bispecific T cell engager (BiTE) molecule; a hormone; a cytokine (e.g., IL-2, insulin, IFN-γ, IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-a), a chemokine (e.g., ΜΙΡ-Ια, ΜΙΡ-Ιβ, MCP-1, MCP-3, and RANTES), a cytotoxin (e.g., Perforin, Granzyme A, and Granzyme B), or a cytokine receptor (e.g., an JL-2 receptor, an IL-7 receptor, an IL-12 receptor, or an IL-15 receptor, and an IL-21 receptor).
In particular embodiments, the cell comprises a polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
In additional embodiments, the polynucleotide is integrated into the IL-lORa gene. In some embodiments, the polynucleotide is a donor repair template integrated into the IL- 1 ORa gene at a DNA double stranded break site introduced by the polypeptide
contemplated herein.
In further embodiments, the polynucleotide is a donor repair template designed to correct one or more loss-of-function mutations in the endogenous IL-lORa gene, and wherein the donor repair template is integrated into the IL-lORa gene at a DNA double stranded break site introduced by the polypeptide contemplated herein.
In further embodiments, IL-lORa expression is maintained, restored or increased and the polynucleotide encodes FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
In particular embodiments, the present invention contemplates, in part, a plurality of cells comprising one or more cells contemplated herein.
In various embodiments, the present invention contemplates, in part, a composition comprising one or more cells contemplated herein.
In certain embodiments, the present invention contemplates, in part, a composition comprising one or more cells contemplated herein and a physiologically acceptable carrier.
In various embodiments, the present invention contemplates, in part, a method of editing a human IL-lORa gene in a cell comprising: introducing a polynucleotide encoding a polypeptide contemplate herein into the cell, wherein expression of the polypeptide creates a double strand break at a target site in a human IL-lORa gene.
In some embodiments, the present invention contemplates, in part, a method of editing a human IL-lORa gene in cell comprising: introducing a polynucleotide encoding a polypeptide contemplated herein into the cell, wherein expression of the polypeptide creates a double strand break at a target site in a human IL-lORa gene, wherein the break is repaired by non-homologous end joining ( HEJ).
In various embodiments, the present invention contemplates, in part, a method of editing a human IL-lORa gene in a cell comprising: introducing a polynucleotide encoding a polypeptide contemplated herein and a donor repair template into the cell, wherein expression of the polypeptide creates a double strand break at a target site in a human IL-lORa gene and the donor repair template is incorporated into the human IL-lORa gene by homology directed repair (HDR) at the site of the double-strand break (DSB).
In additional embodiments, IL-lORa expression is maintained, restored or increased and the polynucleotide encodes FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
In further embodiments, the cell is a hematopoietic cell.
In particular embodiments, the cell is a T cell.
In particular embodiments, the cell is a CD3+, CD4+, and/or CD8+ cell.
In certain embodiments, the cell is an immune effector cell.
In some embodiments, the cell is a cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cells.
In particular embodiments, the cell is a natural killer (NK) cell or natural killer T (NKT) cell.
In additional embodiments, the cell is a regulatory T cell (Treg).
In certain embodiments, the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
In particular embodiments, the polynucleotide encoding the polypeptide is an mRNA.
In additional embodiments, a polynucleotide encoding a 5 -3' exonuclease is introduced into the cell.
In some embodiments, a polynucleotide encoding Trex2 or a biologically active fragment thereof is introduced into the cell. In particular embodiments, the donor repair template encodes a FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
In further embodiments, the donor repair template encodes a wild type copy of the IL- 1 ORa gene or portion thereof.
In further embodiments, the donor repair template encodes an IL-lORa gene or portion thereof comprising one or more mutations compared to the wild type IL-lORa gene.
In particular embodiments, the donor repair template encodes an engineered antigen receptor.
In certain embodiments, the engineered antigen receptor is selected from the group consisting of: an engineered T cell receptor, a chimeric antigen receptor, a DARIC, or a zetakine.
In further embodiments, the donor repair template encodes a bispecific T cell engager (BiTE) molecule; a hormone; a cytokine (e.g., IL-2, insulin, IFN-γ, IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-a), a chemokine (e.g., ΜΙΡ-Ια, ΜΙΡ-Ιβ, MCP-1, MCP-3, and RANTES), a cytotoxin (e.g., Perforin, Granzyme A, and Granzyme B), or a cytokine receptor (e.g., an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, or an IL-15 receptor, and an IL-21 receptor).
In additional embodiments, the donor repair template comprises a 5 ' homology arm homologous to a human IL-lORa gene sequence 5' of the DSB and a 3 ' homology arm homologous to a human IL- 1 ORa gene sequence 3 ' of the D SB .
In particular embodiments, the lengths of the 5' and 3 ' homology arms are independently selected from about 100 bp to about 2500 bp.
In some embodiments, the lengths of the 5' and 3 ' homology arms are independently selected from about 600 bp to about 1500 bp.
In some embodiments, the 5 'homology arm is about 1500 bp and the 3 ' homology arm is about 1000 bp.
In certain embodiments, the 5 'homology arm is about 600 bp and the 3 ' homology arm is about 600 bp.
In particular embodiments, a viral vector is used to introduce the donor repair template into the cell. In additional embodiments, the viral vector is a recombinant adeno-associated viral vector (rAAV) or a retrovirus.
In further embodiments, the rAAV has one or more ITRs from AAV2.
In certain embodiments, the rAAV has a serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.
In additional embodiments, the rAAV has an AAV2 or AAV6 serotype.
In some embodiments, the retrovirus is a lentivirus.
In particular embodiments, the lentivirus is an integrase deficient lentivirus (IDLV). In various embodiments, the present invention contemplates, in part, a method of treating, preventing, or ameliorating at least one symptom of a cancer, GVHD, transplant rejection, infectious disease, autoimmune disease, inflammatory disease, and
immunodeficiency, or condition associated therewith, comprising administering to the subject an effective amount of a composition contemplated herein.
In various embodiments, the present invention contemplates, in part, a method of treating a solid cancer comprising administering to the subj ect an effective amount of a composition contemplated herein.
In further embodiments, the solid cancer comprises liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.
In various embodiments, the present invention contemplates, in part, a method of treating a hemotological malignancy comprising administering to the subject an effective amount of a composition contemplated herein.
In additional embodiments, the hematological malignancy is a leukemia, lymphoma, or multiple myeloma.
In various embodiments, the present invention contemplates, in part, a method of treating an autoimmune disease comprising administering to the subject an effective amount of a composition contemplated herein.
In certain embodiments, the autoimmune disease is associated with a loss-of-function mutation in the IL- 1 ORa gene. In some embodiments, the loss-of-function mutation is a missense mutation, nonsense mutation, or splice site mutation.
In further embodiments, the autoimmune disease is arthritis.
In particular embodiments, the autoimmune disease is inflammatory bowel disease (IBD).
In additional embodiments, the IBD is selected from the group consisting of ulcerative colitis, early onset ulcerative colitis, very early onset ulcerative colitis, pancolitis, Crohn's disease, and neonatal-onset Crohn's disease.
In certain embodiments, the autoimmune disease is associated with a loss-of-function mutation in the IL-lORa gene selected from the group consisting of: W45G; Y64C; W69R; T84I; Y91C; V100G; R101W; R117H; S138G; G141R; I169T; c.537G> A, p.T179T;
g.IVS5+2T>C, c.690_765del, P206X; R262C, and E43 IX.
In various embodiments, the present invention contemplates, in part, a method of treating graft- versus-host disease (GVHD) comprising administering to the subject an effective amount of a composition contemplated herein.
In particular embodiments, the GVHD is associated with a solid organ transplant in the subject.
In some embodiments, the solid organ transplant is selected from the group consisting of: a heart transplant, a lung transplant, a kidney transplant, a pancreas transplant, and a liver transplant.
In various embodiments, the present invention contemplates, in part, a method of preventing graft-versus-host disease (GVHD) while maintaining a graft-versus-leukemia response comprising administering to the subject an effective amount of a composition contemplated herein.
In particular embodiments, the GVHD is associated with a bone marrow transplant in the subject.
BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS
Figure 1 shows the human IL-lORa gene and the location of the homing endonuclease target site within exon 2 (SEQ ID NOs: 60 and 61). Figure 2 shows reprogramming of the I-Onul N-terminal domain (NTD) and C- terminal domain (CTD) against chimeric "half-sites" through three rounds of sorting, followed by fusion of the reprogrammed domains to isolate a fully reprogrammed I-Onul homing endonuclease that cleaves the target site.
Figure 3 shows the initial screening of I-Onul derived homing endonuclease variants for activity against an IL-lORa target site in a chromosomal reporter assay. Figure 3 also shows the refinement of the initially derived I-Onul derived homing endonuclease IL-10Ra.G7 to achieve a more active variants, IL-10Ra.G7.A3 and IL-10Ra.G7.A3.G7.
Figure 4 shows that the IL-10Ra.G7.A3.G7 homing endonuclease has sub-nanomolar affinity properties as measured using a yeast surface display based substrate titration assay.
Figure 5 shows an alignment of IL-10Ra.G7 (SEQ ID NO: 63), IL-10Ra.G7.A3 (SEQ ID NO: 64) and IL-10Ra.G7.A3.G7 (SEQ ID NO: 65) homing endonucleases compared to the wild type I-Onul (SEQ ID NO: 62) homing endonucleases, highlighting non-identical positions.
Figure 6 A shows a schematic of an IL-lORa megaTAL (SEQ ID NO: 67) that targets the IL-lORa gene (SEQ ID NO: 66).
Figure 6B shows a TIDE analysis of the genome editing of IL-lORa megaTAL co- delivered with Trex2 of the IL-lORa target sequence in primary human T cells.
Figure 7A shows a schematic of an AAV donor repair template that targets the IL- lORa gene.
Figure 7B shows that T cells treated with IL-lORa megaTAL and AAV donor repair template undergo HDR.
BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS
SEQ ID NO: 1 is an amino acid sequence of a wild type I-Onul LAGLIDADG homing endonuclease (LHE).
SEQ ID NO: 2 is an amino acid sequence of a wild type I-Onul LHE.
SEQ ID NO: 3 is an amino acid sequence of a biologically active fragment of a wild- type I-Onul LHE. SEQ ID NO: 4 is an amino acid sequence of a biologically active fragment of a wild- type I-Onul LHE.
SEQ ID NO: 5 is an amino acid sequence of a biologically active fragment of a wild- type I-Onul LHE.
SEQ ID NO: 6 is an amino acid sequence of an I-Onul LHE variant reprogrammed to bind and cleave a target site in the human IL-lORa gene.
SEQ ID NO: 7 is an amino acid sequence of an I-Onul LHE variant reprogrammed to bind and cleave a target site in the human IL-lORa gene.
SEQ ID NO: 8 is an amino acid sequence of an I-Onul LHE variant reprogrammed to bind and cleave a target site in the human IL- 1 ORa gene.
SEQ ID NO: 9 is an amino acid sequence of a megaTAL that binds and cleaves a target site in a human IL-lORa gene.
SEQ ID NO: 10 is an amino acid sequence of a megaTAL that binds and cleaves a target site in a human IL-lORa gene.
SEQ ID NO: 11 is an amino acid sequence of a megaTAL that binds and cleaves a target site in a human IL-lORa gene.
SEQ ID NO: 12 is an amino acid sequence of a megaTAL-Trex2 fusion protein that binds and cleaves a target site in a human IL-lORa gene.
SEQ ID NO: 13 is an I-Onul LHE variant target site in a human IL-lORa gene.
SEQ ID NO: 14 is a TALE DNA binding domain target site in a human IL- 1 ORa gene.
SEQ ID NO: 15 is a megaTAL target site in a human IL-lORa gene.
SEQ ID NO: 16 is an I-Onul LHE variant N-terminal domain target site.
SEQ ID NO: 17 is an I-Onul LHE variant C-terminal domain target site.
SEQ ID NO: 18 is a polynucleotide sequence of an I-Onul LHE variant surface display plasmid.
SEQ ID NO: 19 is an mRNA sequence encoding a megaTAL that cleaves a human IL- lORa gene.
SEQ ID NO: 20 is an mRNA sequence encoding murine Trex2.
SEQ ID NO: 21 is an amino acid sequence encoding murine Trex2. SEQ ID NO: 22 is an AAV-based ILlORa pMND-GFP donor repair template.
SEQ ID NOs: 23-33 forth the amino acid sequences of various linkers.
SEQ ID NOs: 34-58 set forth the amino acid sequences of protease cleavage sites and self-cleaving polypeptide cleavage sites.
In the foregoing sequences, X, if present, refers to any amino acid or the absence of an amino acid.
DETAILED DESCRIPTION
A. OVERVIEW
The invention generally relates to, in part, improved genome editing compositions and methods of use thereof. Without wishing to be bound by any particular theory, genome editing compositions contemplated in various embodiments can be used to prevent or treat a cancer, graft- versus-host-disease (GVHD), transplant rejection, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or condition associated therewith, or ameliorates at least one symptom thereof.
Genome editing methods contemplated in particular embodiments are realized, in part, through modification of the IL-10 receptor, IL-lORa. The immunosuppressive effects of IL- 10/IL-lORa signaling in the tumor microenvironment limit the effectiveness of adoptive immunotherapies. Moreover, defects in IL-10 signaling mediated through IL-lORa are associated with a compromised ability to respond to inflammatory disease and autoimmune disorders.
Genome editing compositions and methods contemplated in various embodiments comprise nuclease variants, designed to bind and cleave a target site in the human interleukin 10 receptor 1 alpha (IL-lORa) gene. The nuclease variants contemplated in particular embodiments, can be used to introduce a double-strand break in a target polynucleotide sequence, which may be repaired by non-homologous end joining ( HEJ) in the absence of a polynucleotide template, e.g., a donor repair template, or by homology directed repair (HDR), i.e., homologous recombination, in the presence of a donor repair template. Nuclease variants contemplated in certain embodiments, can also be designed as nickases, which generate single- stranded DNA breaks that can be repaired using the cell's base-excision-repair (BER) machinery or homologous recombination in the presence of a donor repair template. NHEJ is an error-prone process that frequently results in the formation of small insertions and deletions that disrupt gene function. Homologous recombination requires homologous DNA as a template for repair and can be leveraged to create a limitless variety of modifications specified by the introduction of donor DNA containing the desired sequence at the target site, flanked on either side by sequences bearing homology to regions flanking the target site.
In one preferred embodiment, the genome editing compositions contemplated herein comprise a homing endonuclease variant or megaTAL that targets the human IL-lORa gene.
In one preferred embodiment, the genome editing compositions contemplated herein comprise a homing endonuclease variant or megaTAL and an end-processing enzyme, e.g., Trex2.
In various embodiments, genome edited cells are contemplated. The genome edited cells comprise an edited IL-lORa gene, wherein the editing strategy is designed to decrease or eliminate IL-lORa expression or wherein the editing strategy is designed to increase or restore expression of IL-lORa by correcting one or more mutations in the IL-lORa gene.
In one embodiment, the genome editing strategy comprises introducing a
polynucleotide in the IL-lORa gene without disrupting IL-lORa expression. In certain embodiments, the polynucleotide encodes a polypeptide that enhances Treg function.
In various embodiments, a DNA break is generated in a target site of the IL-lORa gene in a T cell or immune effector cell, and NHEJ of the ends of the cleaved genomic sequence may result in a cell with little or no IL-lORa expression, and preferably a T cell that lacks or substantially lacks functional IL-lORa expression, e.g., lacks the ability to increase T cell exhaustion and to inhibit expression of MHC class Π molecules, costimulatory molecules, and proinflammatory cytokines. Without wishing to be bound by any particular theory, T cells that lack IL-lORa expression are more resistant to immunosuppression and T cell exhaustion, and thus, are more therapeutically efficacious.
In various other embodiments, a donor template for repair of the cleaved IL-lORa genomic sequence is provided. The IL-lORa gene is repaired with the sequence of the template by homologous recombination at the DNA break-site. In particular embodiments, the repair template comprises a polynucleotide sequence that is different from a targeted genomic sequence. In particular embodiments, the repair template comprises a polynucleotide encoding an IL-lORa sequence that restores IL-lORa function.
In various other embodiments, a donor template encoding a modified IL-lORa polypeptide may be used to repair the cleaved IL-lORa genomic sequence. The IL-lORa gene is repaired with the sequence of the template by homologous recombination at the DNA break- site. In particular embodiments, the repair template comprises a polynucleotide encoding an IL-lORa sequence that modifies IL-lORa function, by increasing or decreasing receptor signaling, e.g., by modifying the affinity of IL-lORa for its cognate ligand IL-10.
In various embodiments, a DNA break is generated in a target site of the IL- 1 ORa gene in a T cell or regulatory T cell (Treg), and donor template encoding a polypeptide that enhances Treg function is introduced into the IL-lORa gene at the double-stranded DNA break, without disrupting expression of IL-lORa. Without wishing to be bound by any particular theory, it is contemplated that T cells or Tregs that comprise an edited IL-lORa gene comprising a polynucleotide encoding a polypeptide that enhances Treg function expressed from the IL- 10Ra promoter or an exogenous promoter inserted into the IL-lORa gene are more stable Tregs that are more therapeutically efficacious in maintaining graft-versus-leukemia (GVL) activity, e.g., post bone marrow or solid organ transplants, preventing transplant rejections, e.g., from bone marrow or solid organ transplants, treating graft-versus-host-disease (GVHD), e.g., resulting from bone marrow or solid organ transplants, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or a condition associated therewith.
In preferred embodiments, the genome editing compositions and methods
contemplated herein are used to edit the human IL-lORa gene.
Accordingly, the methods and compositions contemplated herein represent a quantum improvement compared to existing adoptive cell therapies.
The practice of the particular embodiments will employ, unless indicated specifically to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that are within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See e.g., Sambrook, etal., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al, Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al, Molecular Cloning: A Laboratory Manual (1982); Ausubel et al, Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A
Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1998) Current Protocols in Immunology Q. E. Coligan, A. M. Kmisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as monographs in journals such as Advances in
Immunology.
B. DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below.
The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.
The use of the alternative {e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.
The term "and/or" should be understood to mean either one, or both of the alternatives.
As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term "about" or "approximately" refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%>, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
In one embodiment, a range, e.g., 1 to 5, about 1 to 5, or about 1 to about 5, refers to each numerical value encompassed by the range. For example, in one non-limiting and merely illustrative embodiment, the range "1 to 5" is equivalent to the expression 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.
As used herein, the term "substantially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is 80%>, 85%>, 90%>, 91%>, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, "substantially the same" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that produces an effect, e.g., a physiological effect, that is approximately the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
Throughout this specification, unless the context requires otherwise, the words
"comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that no other elements are present that materially affect the activity or action of the listed elements. Reference throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," or "a further embodiment" or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment.
Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in a particular embodiment.
The term "ex vivo" refers generally to activities that take place outside an organism, such as experimentation or measurements done in or on living tissue in an artificial environment outside the organism, preferably with minimum alteration of the natural conditions. In particular embodiments, "ex vivo" procedures involve living cells or tissues taken from an organism and cultured or modulated in a laboratory apparatus, usually under sterile conditions, and typically for a few hours or up to about 24 hours, but including up to 48 or 72 hours, depending on the circumstances. In certain embodiments, such tissues or cells can be collected and frozen, and later thawed for ex vivo treatment. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissue are typically considered to be "in vitro " though in certain embodiments, this term can be used interchangeably with ex vivo.
The term "in vivo" refers generally to activities that take place inside an organism. In one embodiment, cellular genomes are engineered, edited, or modified in vivo.
By "enhance" or "promote" or "increase" or "expand" or "potentiate" refers generally to the ability of a nuclease variant, genome editing composition, or genome edited cell contemplated herein to produce, elicit, or cause a greater response {i.e., physiological response) compared to the response caused by either vehicle or control. A measurable response may include an increase in catalytic activity, binding affinity, persistence, in cytolytic activity, and/or an increase in proinflammatory cytokines, among others apparent from the
understanding in the art and the description herein. An "increased" or "enhanced" amount is typically a "statistically significant" amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or control.
By "decrease" or "lower" or "lessen" or "reduce" or "abate" or "ablate" or "inhibit" or
"dampen" refers generally to the ability of a nuclease variant, genome editing composition, or genome edited cell contemplated herein to produce, elicit, or cause a lesser response (i.e., physiological response) compared to the response caused by either vehicle or control. A measurable response may include a decrease in off-target binding affinity, off- target cleavage specificity, anti-inflammatory cytokine production and/or secretion, and the like. A "decrease" or "reduced" amount is typically a "statistically significant" amount, and may include an decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle, or control.
By "maintain," or "preserve," or "maintenance," or "no change," or "no substantial change," or "no substantial decrease" refers generally to the ability of a nuclease variant, genome editing composition, or genome edited cell contemplated herein to produce, elicit, or cause a substantially similar or comparable physiological response (i.e., downstream effects) in as compared to the response caused by either vehicle or control. A comparable response is one that is not significantly different or measurable different from the reference response.
The terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targets" as used herein, describe binding of one molecule to another, e.g., DNA binding domain of a polypeptide binding to DNA, at greater binding affinity than background binding. A binding domain "specifically binds" to a target site if it binds to or associates with a target site with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of l/M) of, for example, greater than or equal to about 105 M"1. In certain embodiments, a binding domain binds to a target site with a Ka greater than or equal to about 106 M"1, 107 M"1, 108 M"1, 109 M"1, 1010 M"1, 1011 M"1, 1012 M" 1, or 1013 M"1. "High affinity" binding domains refers to those binding domains with a Ka of at least 107 M"1, at least 108 M"1, at least 109 M"1, at least 1010 M"1, at least 1011 M"1, at least 1012 M"1, at least 1013 M"1, or greater.
Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10"5 M to 10"13 M, or less). Affinities of nuclease variants comprising one or more DNA binding domains for DNA target sites contemplated in particular embodiments can be readily determined using conventional techniques, e.g., yeast cell surface display, or by binding association, or displacement assays using labeled ligands.
In one embodiment, the affinity of specific binding is about 2 times greater than background binding, about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.
The terms "selectively binds" or "selectively bound" or "selectively binding" or "selectively targets" and describe preferential binding of one molecule to a target molecule (on- target binding) in the presence of a plurality of off-target molecules. In particular
embodiments, an HE or megaTAL selectively binds an on-target DNA binding site about 5, 10, 15, 20, 25, 50, 100, or 1000 times more frequently than the HE or megaTAL binds an off-target DNA target binding site.
"On-target" refers to a target site sequence.
"Off-target" refers to a sequence similar to but not identical to a target site sequence.
A "target site" or "target sequence" is a chromosomal or extrachromosomal nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind and/or cleave, provided sufficient conditions for binding and/or cleavage exist. When referring to a polynucleotide sequence or SEQ ID NO. that references only one strand of a target site or target sequence, it would be understood that the target site or target sequence bound and/or cleaved by a nuclease variant is double-stranded and comprises the reference sequence and its complement. In a preferred embodiment, the target site is a sequence in a human IL-lORa gene. "Recombination" refers to a process of exchange of genetic information between two polynucleotides, including but not limited to, donor capture by non-homologous end joining ( HEJ) and homologous recombination. For the purposes of this disclosure, "homologous recombination (HR)" refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells via homology-directed repair (HDR) mechanisms. This process requires nucleotide sequence homology, uses a "donor" molecule as a template to repair a "target" molecule (i.e., the one that experienced the double-strand break), and is variously known as "non-crossover gene conversion" or "short tract gene conversion," because it leads to the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer can 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 is used to resynthesize genetic information that will become part of the target, and/or related processes. Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.
"NHEJ" or "non-homologous end joining" refers to the resolution of a double-strand break in the absence of a donor repair template or homologous sequence. NHEJ can result in insertions and deletions at the site of the break. NHEJ is mediated by several sub-pathways, each of which has distinct mutational consequences. The classical NHEJ pathway (cNHEJ) requires the KU/DNA-PKcs/Lig4/XRCC4 complex, ligates ends back together with minimal processing and often leads to precise repair of the break. Alternative NHEJ pathways
(altNHEJ) also are active in resolving dsDNA breaks, but these pathways are considerably more mutagenic and often result in imprecise repair of the break marked by insertions and deletions. While not wishing to be bound to any particular theory, it is contemplated that modification of dsDNA breaks by end-processing enzymes, such as, for example,
exonucleases, e.g., Trex2, may bias repair towards an altNHEJ pathway.
"Cleavage" refers to the breakage of the covalent backbone of a DNA molecule.
Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double- stranded cleavage are possible. Double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, polypeptides and nuclease variants, e.g., homing endonuclease variants, megaTALs, etc. contemplated herein are used for targeted double-stranded DNA cleavage. Endonuclease cleavage recognition sites may be on either DNA strand.
An "exogenous" molecule is a molecule that is not normally present in a cell, but that is introduced into a cell by one or more genetic, biochemical or other methods. Exemplary exogenous molecules include, but are not limited to small organic molecules, protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.
Methods for the introduction of exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, biopolymer nanoparticle, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.
An "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. Additional endogenous molecules can include proteins.
A "gene," refers to a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. A gene includes, but is not limited to, promoter sequences, enhancers, silencers, insulators, boundary elements, terminators, polyadenylation sequences, post-transcription response elements, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, replication origins, matrix attachment sites, and locus control regions.
"Gene expression" refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
As used herein, the term "genetically engineered" or "genetically modified" refers to the chromosomal or extrachromosomal addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. Genetic modifications may be targeted or non- targeted to a particular site in a cell's genome. In one embodiment, genetic modification is site specific. In one embodiment, genetic modification is not site specific.
As used herein, the term "genome editing" refers to the substitution, deletion, and/or introduction of genetic material at a target site in the cell's genome, which restores, corrects, disrupts, and/or modifies expression of a gene or gene product. Genome editing contemplated in particular embodiments comprises introducing one or more nuclease variants into a cell to generate DNA lesions at or proximal to a target site in the cell's genome, optionally in the presence of a donor repair template.
As used herein, the term "gene therapy" refers to the introduction of extra genetic material into the total genetic material in a cell that restores, corrects, or modifies expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide. In particular embodiments, introduction of genetic material into the cell's genome by genome editing that restores, corrects, disrupts, or modifies expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide is considered gene therapy.
An "immune disorder" refers to a disease that evokes a response from the immune system. In particular embodiments, the term "immune disorder" refers to a cancer, graft- versus-host disease, an autoimmune disease, or an immunodeficiency. In one embodiment, immune disorders encompasses infectious disease.
As used herein, the term "cancer" relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues.
As used herein, the term "malignant" refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood). As used herein, the term "metastasize" refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a "metastatic tumor" or a "metastasis." The metastatic tumor contains cells that are like those in the original (primary) tumor.
As used herein, the term "benign" or "non-malignant" refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
A "cancer cell" or "tumor cell" refers to an individual cell of a cancerous growth or tissue. A tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancers form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor.
" Graft- versus-host disease" or "GVHD" refers complications that can occur after cell, tissue, or solid organ transplant. GVHD can occur after a stem cell or bone marrow transplant in which the transplanted donor cells attack the transplant recipient's body. Acute GVHD in humans takes place within about 60 days post-transplantation and results in damage to the skin, liver, and gut by the action of cytolytic lymphocytes. Chronic GVHD occurs later and is a systemic autoimmune disease that affects primarily the skin, resulting in the polyclonal activation of B cells and the hyperproduction of Ig and autoantibodies. Solid-organ transplant graft-versus-host disease (SOT-GVHD) occurs in two forms. The more common type is antibody mediated, wherein antibodies from a donor with blood type O attack a recipient's red blood cells in recipients with blood type A, B, or AB, leading to mild transient, hemolytic anemias. The second form of SOT-GVHD is a cellular type associated with high mortality, wherein donor-derived T cells produce an immunological attack against immunologically disparate host tissue, most often in the skin, liver, gastrointestinal tract, and bone marrow, leading to complications in these organs. "Graft- versus-leukemia" or "GVL" refer to an immune response to a person's leukemia cells by immune cells present in a donor's transplanted tissue, such as bone marrow or peripheral blood.
An "autoimmune disease" refers to a disease in which the body produces an immunogenic (i.e., immune system) response to some constituent of its own tissue. In other words, the immune system loses its ability to recognize some tissue or system within the body as "self and targets and attacks it as if it were foreign. Illustrative examples of autoimmune diseases include, but are not limited to: arthritis, inflammatory bowel disease, Hashimoto's thyroiditis, Grave's disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti -immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like.
An "immunodeficiency" means the state of a patient whose immune system has been compromised by disease or by administration of chemicals. This condition makes the system deficient in the number and type of blood cells needed to defend against a foreign substance. Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immunodeficiency syndrome), SCID (severe combined immunodeficiency disease), selective IgA deficiency, common variable immunodeficiency, X-linked agammaglobulinemia, chronic granulomatous disease, hyper-IgM syndrome, Wiskott-Aldrich Syndrome (WAS), and diabetes.
An "infectious disease" refers to a disease that can be transmitted from person to person or from organism to organism, and is caused by a microbial or viral agent (e.g., common cold). Infectious diseases are known in the art and include, for example, hepatitis, sexually transmitted diseases (e.g., Chlamydia, gonorrhea), tuberculosis, HIV/AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.
As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits a symptom of an immune disorder that can be treated with the nuclease variants, genome editing compositions, gene therapy vectors, genome editing vectors, genome edited cells, and methods contemplated elsewhere herein. Suitable subjects (e.g., patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human subjects, are included. Typical subjects include human patients that have, have been diagnosed with, or are at risk of having an immune disorder.
As used herein, the term "patient" refers to a subject that has been diagnosed with an immune disorder that can be treated with the nuclease variants, genome editing compositions, gene therapy vectors, genome editing vectors, genome edited cells, and methods contemplated elsewhere herein.
As used herein "treatment" or "treating," includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. Treatment can optionally involve delaying of the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.
As used herein, "prevent," and similar words such as "prevention," "prevented," "preventing" etc. , indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition, e.g., cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.
As used herein, the phrase "ameliorating at least one symptom of refers to decreasing one or more symptoms of the disease or condition for which the subject is being treated, e.g., cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and
immunodeficiency. In particular embodiments, the disease or condition being treated is a cancer, wherein the one or more symptoms ameliorated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, distended or painful abdomen (due to enlarged abdominal organs), bone or joint pain, fractures, unplanned weight loss, poor appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function). As used herein, the term "amount" refers to "an amount effective" or "an effective amount" of a nuclease variant, genome editing composition, or genome edited cell sufficient to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.
A "prophylactically effective amount" refers to an amount of a nuclease variant, genome editing composition, or genome edited cell sufficient to achieve the desired
prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.
A "therapeutically effective amount" of a nuclease variant, genome editing
composition, or genome edited cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).
C. NUCLEASE VARIANTS
Nuclease variants contemplated in particular embodiments herein are suitable for genome editing a target site in the IL-lORa gene and comprise one or more DNA binding domains and one or more DNA cleavage domains (e.g., one or more endonuclease and/or exonuclease domains), and optionally, one or more linkers contemplated herein. The terms "reprogrammed nuclease," "engineered nuclease," or "nuclease variant" are used
interchangeably and refer to a nuclease comprising one or more DNA binding domains and one or more DNA cleavage domains, wherein the nuclease has been designed and/or modified from a parental or naturally occurring nuclease, to bind and cleave a double-stranded DNA target sequence in an IL-lORa gene. In particular embodiments, a nuclease variant binds and cleaves a target sequence in exon 2 of an IL-lORa gene, preferably at SEQ ID NO: 13 in exon 2 of an IL-lORa gene, and more preferably at the sequence "ATTC" in SEQ ID NO: 13 in exon 2 of an IL-lORa gene.
The nuclease variant may be designed and/or modified from a naturally occurring nuclease or from a previous nuclease variant. Nuclease variants contemplated in particular embodiments may further comprise one or more additional functional domains, e.g., an end- processing enzymatic domain of an end-processing enzyme that exhibits 5 '-3' exonuclease, 5'- 3' alkaline exonuclease, 3 '-5 'exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerases or template-independent DNA polymerase activity.
Illustrative examples of nuclease variants that bind and cleave a target sequence in the
IL-lORa gene include, but are not limited to homing endonuclease (meganuclease) variants and megaTALs.
1. HOMING ENDONUCLEASE (MEGANUCLEASE) VARIANTS
In various embodiments, a homing endonuclease or meganuclease is reprogrammed to introduce a double-strand break (DSB) in a target site in an IL-lORa gene. In particular embodiments, a homing endonuclease variant introduces a double strand break in exon 2 of an IL-lORa gene, preferably at SEQ ID NO: 13 in exon 2 of an IL-lORa gene, and more preferably at the sequence "ATTC" in SEQ ID NO: 13 in exon 2 of an IL-lORa gene.
"Homing endonuclease" and "meganuclease" are used interchangeably and refer to naturally- occurring homing endonucl eases that recognize 12-45 base-pair cleavage sites and are commonly grouped into five families based on sequence and structure motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D/E)XK.
A "reference homing endonuclease" or "reference meganuclease" refers to a wild type homing endonuclease or a homing endonuclease found in nature. In one embodiment, a "reference homing endonuclease" refers to a wild type homing endonuclease that has been modified to increase basal activity.
An "engineered homing endonuclease," "reprogrammed homing endonuclease," "homing endonuclease variant," "engineered meganuclease," "reprogrammed meganuclease," or "meganuclease variant" refers to a homing endonuclease comprising one or more DNA binding domains and one or more DNA cleavage domains, wherein the homing endonuclease has been designed and/or modified from a parental or naturally occurring homing
endonuclease, to bind and cleave a DNA target sequence in an IL-lORa gene. The homing endonuclease variant may be designed and/or modified from a naturally occurring homing endonuclease or from another homing endonuclease variant. Homing endonuclease variants contemplated in particular embodiments may further comprise one or more additional functional domains, e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 '-3' exonuclease, 5 '-3' alkaline exonuclease, 3 '-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template dependent DNA polymerase or template-independent DNA polymerase activity.
Homing endonuclease (HE) variants do not exist in nature and can be obtained by recombinant DNA technology or by random mutagenesis. HE variants may be obtained by making one or more amino acid alterations, e.g., mutating, substituting, adding, or deleting one or more amino acids, in a naturally occurring HE or HE variant. In particular embodiments, a HE variant comprises one or more amino acid alterations to the DNA recognition interface.
HE variants contemplated in particular embodiments may further comprise one or more linkers and/or additional functional domains, e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 '-3' exonuclease, 5 '-3' alkaline exonuclease, 3 '-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA
polymerase or template-independent DNA polymerase activity. In particular embodiments, HE variants are introduced into a T cell with an end-processing enzyme that exhibits 5 '-3 ' exonuclease, 5 '-3' alkaline exonuclease, 3 '-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerase or template-independent DNA polymerase activity. The HE variant and 3' processing enzyme may be introduced separately, e.g., in different vectors or separate mRNAs, or together, e.g., as a fusion protein, or in a polycistronic construct separated by a viral self-cleaving peptide or an IRES element.
A "DNA recognition interface" refers to the HE amino acid residues that interact with nucleic acid target bases as well as those residues that are adjacent. For each HE, the DNA recognition interface comprises an extensive network of side chain-to-side chain and side chain-to-DNA contacts, most of which is necessarily unique to recognize a particular nucleic acid target sequence. Thus, the amino acid sequence of the DNA recognition interface corresponding to a particular nucleic acid sequence varies significantly and is a feature of any natural or HE variant. By way of non-limiting example, a HE variant contemplated in particular embodiments may be derived by constructing libraries of HE variants in which one or more amino acid residues localized in the DNA recognition interface of the natural HE (or a previously generated HE variant) are varied. The libraries may be screened for target cleavage activity against each predicted IL-lORa target site using cleavage assays (see e.g., Jarjour et al, 2009. Nuc. Acids Res. 37(20): 6871-6880).
LAGLIDADG homing endonucleases (LHE) are the most well studied family of homing endonucleases, are primarily encoded in archaea and in organellar DNA in green algae and fungi, and display the highest overall DNA recognition specificity. LHEs comprise one or two LAGLIDADG catalytic motifs per protein chain and function as homodimers or single chain monomers, respectively. Structural studies of LAGLIDADG proteins identified a highly conserved core structure (Stoddard 2005), characterized by an αββαββα fold, with the
LAGLIDADG motif belonging to the first helix of this fold. The highly efficient and specific cleavage of LHE' s represent a protein scaffold to derive novel, highly specific endonucleases. However, engineering LHEs to bind and cleave a non-natural or non-canonical target site requires selection of the appropriate LHE scaffold, examination of the target locus, selection of putative target sites, and extensive alteration of the LHE to alter its DNA contact points and cleavage specificity, at up to two-thirds of the base-pair positions in a target site.
In one embodiment, LHEs from which reprogrammed LHEs or LHE variants may be designed include, but are not limited to I-Crel and I-Scel.
Illustrative examples of LHEs from which reprogrammed LHEs or LHE variants may be designed include, but are not limited to I-AabMI, I-AaeMI, I-Anil, I-ApaMI, I-CapIII, I- CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I- EjeMI, I-GpeMI, I-Gpil, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-Ltrll, I-Ltrl, I-LtrWI, I- MpeMI, I-MveMI, I-Ncrll, I-Ncrl, I-NcrMI, I-OheMI, I-Onul, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, and I- Vdil41I. In one embodiment, the reprogrammed LHE or LHE variant is selected from the group consisting of: an I-CpaMI variant, an I-HjeMI variant, an I-Onul variant, an I-PanMI variant, and an I-SmaMI variant.
In one embodiment, the reprogrammed LHE or LHE variant is an I-Onul variant. See e.g., SEQ ID NOs: 6-8.
In one embodiment, reprogrammed I-Onul LHEs or I-Onul variants targeting the IL- 10Ra gene were generated from a natural I-Onul or biologically active fragment thereof (SEQ ID NOs: 1-5). In a preferred embodiment, reprogrammed I-Onul LHEs or I-Onul variants targeting the human IL-lORa gene were generated from an existing I-Onul variant. In one embodiment, reprogrammed I-Onul LHEs were generated against a human IL- 1 ORa gene target site set forth in SEQ ID NO: 13.
In a particular embodiment, the reprogrammed I-Onul LHE or I-Onul variant that binds and cleaves a human IL-lORa gene comprises one or more amino acid substitutions in the DNA recognition interface. In particular embodiments, the I-Onul LHE that binds and cleaves a human IL-lORa gene comprises at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the DNA recognition interface of I-Onul (Taekuchi et al. 2011. Proc Natl Acad Sci U. S. A. 2011 Aug 9; 108(32): 13077-13082) or an I-Onul LHE variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
In one embodiment, the I-Onul LHE that binds and cleaves a human IL-lORa gene comprises at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 99% sequence identity with the DNA recognition interface of I-Onul (Taekuchi et al. 2011. Proc Natl Acad Sci U. S. A. 2011 Aug 9; 108(32): 13077-13082) or an l- Onul LHE variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof. In a particular embodiment, an I-Onul LHE variant that binds and cleaves a human IL- lORa gene comprises one or more amino acid substitutions or modifications in the DNA recognition interface of an I-Onul as set forth in any one of SEQ ID NOs: 1-8.
In a particular embodiment, an I-Onul LHE variant that binds and cleaves a human IL- 10Ra gene comprises one or more amino acid substitutions or modifications in the DNA recognition interface, particularly in the subdomains situated from positions 24-50, 68 to 82, 180 to 203 and 223 to 240 of I-Onul (SEQ ID NOs: 1-5) or an I-Onul variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
In a particular embodiment, an I-Onul LHE that binds and cleaves a human IL- 1 ORa gene comprises one or more amino acid substitutions or modifications in the DNA recognition interface at amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of I-Onul (SEQ ID NOs: 1-5) or an I-Onul variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
In a particular embodiment, an I-Onul LHE variant that binds and cleaves a human IL- 10Ra gene comprises 5, 10, 15, 20, 25, 30, 35, or 40 or more amino acid substitutions or modifications in the DNA recognition interface, particularly in the subdomains situated from positions 24-50, 68 to 82, 180 to 203 and 223 to 240 of I-Onul (SEQ ID NOs: 1-5) or an I- Onul variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
In a particular embodiment, an I-Onul LHE variant that binds and cleaves a human IL- 10Ra gene comprises 5, 10, 15, 20, 25, 30, 35, or 40 or more amino acid substitutions or modifications in the DNA recognition interface at amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of (I-Onul SEQ ID NOs: 1-5) or an I- Onul variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof. In one embodiment, an I-Onul LHE variant that binds and cleaves a human IL-lORa gene comprises one or more amino acid substitutions or modifications at additional positions situated anywhere within the entire I-Onul sequence. The residues which may be substituted and/or modified include but are not limited to amino acids that contact the nucleic acid target or that interact with the nucleic acid backbone or with the nucleotide bases, directly or via a water molecule. In one non-limiting example, an I-Onul LHE variant contemplated herein that binds and cleaves a human IL-lORa gene comprises one or more substitutions and/or modifications, preferably at least 5, preferably at least 10, preferably at least 15, preferably at least 20, more preferably at least 25, more preferably at least 30, even more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions in at least one position selected from the position group consisting of positions: 24, 26, 28, 30, 32, 34, 36, 37, 38, 40, 41, 42, 44, 46, 48, 59, 70, 72, 75, 78, 80, 82, 138, 143, 145, 159, 168, 180, 182, 184, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 207, 223, 225, 227, 228, 229, 232, 236, 238, and 240, of I-Onul (SEQ ID NOs: 1-5) or an I-Onul variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
In a particular embodiment, an I-Onul LHE variant that binds and cleaves a human IL- lORa gene comprises at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46V, Q46I, T48G, N59S, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180H, C180Y, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K, and V238I, or an I-Onul variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
In another particular embodiment, an I-Onul LHE variant that binds and cleaves a human IL-lORa gene comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46V, T48G, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, S159P, F168L, C180H, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, K229A, F232K, D236K, and V238I or an I-Onul variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
In yet another particular embodiment, an I-Onul LHE variant that binds and cleaves a human IL-lORa gene comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46I, T48G, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180H, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K, and V238I, or an I-Onul variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
In yet another particular embodiment, an I-Onul LHE variant that binds and cleaves a human IL-lORa gene comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46I, T48G, N59S, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180Y, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K, and V238I, or an I-Onul variant as set forth in any one of SEQ ID NOs: 6-8, biologically active fragments thereof, and/or further variants thereof.
In particular embodiments, an I-Onul LHE variant that binds and cleaves a human IL- 10Ra gene comprises an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 6-8, or a biologically active fragment thereof.
In particular embodiments, an I-Onul LHE variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 6-8, or a biologically active fragment thereof.
In particular embodiments, an I-Onul LHE variant comprises an amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.
In particular embodiments, an I-Onul LHE variant comprises an amino acid sequence set forth in SEQ ID NO: 7, or a biologically active fragment thereof.
In particular embodiments, an I-Onul LHE variant comprises an amino acid sequence set forth in SEQ ID NO: 8, or a biologically active fragment thereof. 2. MEGATALS
In various embodiments, a megaTAL comprising a homing endonuclease variant is reprogrammed to introduce a double-strand break (DSB) in a target site in an IL-lORa gene. In particular embodiments, a homing endonuclease variant is reprogrammed to introduce a DSB in a target site in exon 2 of an IL-lORa gene, preferably at SEQ ID NO: 13 in exon 2 of an IL- 10Ra gene, and more preferably at the sequence "ATTC" in SEQ ID NO: 13 in exon 2 of an IL-lORa gene.
A "megaTAL" refers to a polypeptide comprising a TALE DNA binding domain and a homing endonuclease variant that binds and cleaves a DNA target sequence in an IL-lORa gene, and optionally comprises one or more linkers and/or additional functional domains, e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 '-3' exonuclease, 5 '-3' alkaline exonuclease, 3 '-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase or template-independent DNA polymerase activity.
In particular embodiments, a megaTAL can be introduced into a cell along with an end- processing enzyme that exhibits 5 '-3 ' exonuclease, 5 '-3 ' alkaline exonuclease, 3 '-5 ' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA
polymerase, or template-independent DNA polymerase activity. The megaTAL and 3 ' processing enzyme may be introduced separately, e.g., in different vectors or separate mRNAs, or together, e.g., as a fusion protein, or in a polycistronic construct separated by a viral self- cleaving peptide or an IRES element.
A "TALE DNA binding domain" is the DNA binding portion of transcription activator-like effectors (TALE or TAL-effectors), which mimics plant transcriptional activators to manipulate the plant transcriptome (see e.g., Kay etal., 2007. Science 318:648-651). TALE DNA binding domains contemplated in particular embodiments are engineered de novo or from naturally occurring TALEs, e.g., AvrBs3 from Xanthomonas campestris pv. vesicatoria, Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonas euvesicatoria, and
Xanthomonas oryzae and brgl 1 and hpxl7 from Ralstonia solanacearum. Illustrative examples of TALE proteins for deriving and designing DNA binding domains are disclosed in U.S. Patent No. 9,017,967, and references cited therein, all of which are incorporated herein by reference in their entireties.
In particular embodiments, a megaTAL comprises a TALE DNA binding domain comprising one or more repeat units that are involved in binding of the TALE DNA binding domain to its corresponding target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length. Each TALE DNA binding domain repeat unit includes 1 or 2 DNA-binding residues making up the Repeat Variable Di-Residue (RVD), typically at positions 12 and/or 13 of the repeat. The natural (canonical) code for DNA recognition of these TALE DNA binding domains has been determined such that an HD sequence at positions 12 and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, NN binds to G or A, and NG binds to T. In certain embodiments, non-canonical (atypical) RVDs are contemplated.
Illustrative examples of non-canonical RVDs suitable for use in particular megaTALs contemplated in particular embodiments include, but are not limited to HH, KH, NH, NK, NQ, RH, RN, SS, NN, SN, KN for recognition of guanine (G); NI, KI, RI, HI, SI for recognition of adenine (A); NG, HG, KG, RG for recognition of thymine (T); RD, SD, HD, ND, KD, YG for recognition of cytosine (C); NV, HN for recognition of A or G; and H*, HA, KA, N*, NA, NC, NS, RA, S*for recognition of A or T or G or C, wherein (*) means that the amino acid at position 13 is absent. Additional illustrative examples of RVDs suitable for use in particular megaTALs contemplated in particular embodiments further include those disclosed in U. S. Patent No. 8,614,092, which is incorporated herein by reference in its entirety.
In particular embodiments, a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 3 to 30 repeat units. In certain embodiments, a megaTAL comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 TALE DNA binding domain repeat units. In a preferred embodiment, a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 5-15 repeat units, more preferably 7-15 repeat units, more preferably 9-15 repeat units, and more preferably 9, 10, 11, 12, 13, 14, or 15 repeat units.
In particular embodiments, a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 3 to 30 repeat units and an additional single truncated TALE repeat unit comprising 20 amino acids located at the C-terminus of a set of TALE repeat units, i.e., an additional C-terminal half-TALE DNA binding domain repeat unit (amino acids -20 to -1 of the C-cap disclosed elsewhere herein, infra). Thus, in particular embodiments, a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 3.5 to 30.5 repeat units. In certain embodiments, a megaTAL comprises 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, 15.5, 16.5, 17.5, 18.5, 19.5, 20.5, 21.5, 22.5, 23.5, 24.5, 25.5, 26.5, 27.5, 28.5, 29.5, or 30.5 TALE DNA binding domain repeat units. In a preferred embodiment, a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 5.5-15.5 repeat units, more preferably 7.5-15.5 repeat units, more preferably 9.5-15.5 repeat units, and more preferably 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, or 15.5 repeat units.
In particular embodiments, a megaTAL comprises a TAL effector architecture comprising an "N-terminal domain (NTD)" polypeptide, one or more TALE repeat domains/units, a "C-terminal domain (CTD)" polypeptide, and a homing endonuclease variant. In some embodiments, the NTD, TALE repeats, and/or CTD domains are from the same species. In other embodiments, one or more of the NTD, TALE repeats, and/or CTD domains are from different species.
As used herein, the term "N-terminal domain (NTD)" polypeptide refers to the sequence that flanks the N-terminal portion or fragment of a naturally occurring TALE DNA binding domain. The NTD sequence, if present, may be of any length as long as the TALE DNA binding domain repeat units retain the ability to bind DNA. In particular embodiments, the NTD polypeptide comprises at least 120 to at least 140 or more amino acids N-terminal to the TALE DNA binding domain (0 is amino acid 1 of the most N-terminal repeat unit). In particular embodiments, the NTD polypeptide comprises at least about 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or at least 140 amino acids N-terminal to the TALE DNA binding domain. In one embodiment, a megaTAL contemplated herein comprises an NTD polypeptide of at least about amino acids +1 to +122 to at least about +1 to +137 of aXanthomonas TALE protein (0 is amino acid 1 of the most N- terminal repeat unit). In particular embodiments, the NTD polypeptide comprises at least about 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137 amino acids N-terminal to the TALE DNA binding domain of aXanthomonas TALE protein. In one embodiment, a megaTAL contemplated herein comprises an NTD polypeptide of at least amino acids +1 to +121 of aRalstonia TALE protein (0 is amino acid 1 of the most N-terminal repeat unit). In particular embodiments, the NTD polypeptide comprises at least about 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137 amino acids N-terminal to the TALE DNA binding domain of a Ralstonia TALE protein.
As used herein, the term "C-terminal domain (CTD)" polypeptide refers to the sequence that flanks the C-terminal portion or fragment of a naturally occurring TALE DNA binding domain. The CTD sequence, if present, may be of any length as long as the TALE DNA binding domain repeat units retain the ability to bind DNA. In particular embodiments, the CTD polypeptide comprises at least 20 to at least 85 or more amino acids C-terminal to the last full repeat of the TALE DNA binding domain (the first 20 amino acids are the half-repeat unit C-terminal to the last C-terminal full repeat unit). In particular embodiments, the CTD polypeptide comprises at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 443, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 76, 77, 78, 79, 80, 81, 82, 83, 84, or at least 85 amino acids C-terminal to the last full repeat of the TALE DNA binding domain. In one embodiment, a megaTAL contemplated herein comprises a CTD polypeptide of at least about amino acids -20 to -1 of a Xanthomonas TALE protein (-20 is amino acid 1 of a half- repeat unit C-terminal to the last C-terminal full repeat unit). In particular embodiments, the CTD polypeptide comprises at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids C-terminal to the last full repeat of the TALE DNA binding domain of a Xanthomonas TALE protein. In one embodiment, a megaTAL contemplated herein comprises a CTD polypeptide of at least about amino acids -20 to -1 of aRalstonia TALE protein (-20 is amino acid 1 of a half-repeat unit C-terminal to the last C-terminal full repeat unit). In particular embodiments, the CTD polypeptide comprises at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids C-terminal to the last full repeat of the TALE DNA binding domain of a Ralstonia TALE protein.
In particular embodiments, a megaTAL contemplated herein, comprises a fusion polypeptide comprising a TALE DNA binding domain engineered to bind a target sequence, a homing endonuclease reprogrammed to bind and cleave a target sequence, and optionally an NTD and/or CTD polypeptide, optionally joined to each other with one or more linker polypeptides contemplated elsewhere herein. Without wishing to be bound by any particular theory, it is contemplated that a megaTAL comprising TALE DNA binding domain, and optionally an NTD and/or CTD polypeptide is fused to a linker polypeptide which is further fused to a homing endonuclease variant. Thus, the TALE DNA binding domain binds a DNA target sequence that is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides away from the target sequence bound by the DNA binding domain of the homing endonuclease variant. In this way, the megaTALs contemplated herein, increase the specificity and efficiency of genome editing.
In one embodiment, a megaTAL comprises a homing endonuclease variant and a
TALE DNA binding domain that binds a nucleotide sequence that is within about 4, 5, or 6 nucleotides, preferably, 6 nucleotides upstream of the binding site of the reprogrammed homing endonuclease.
In one embodiment, a megaTAL comprises a homing endonuclease variant and a TALE DNA binding domain that binds the nucleotide sequence set forth in SEQ ID NO: 14, which is 6 nucleotides upstream of the nucleotide sequence bound and cleaved by the homing endonuclease variant (SEQ ID NO: 13). In preferred embodiments, the megaTAL target sequence is SEQ ID NO: 15.
In particular embodiments, a megaTAL contemplated herein, comprises one or more TALE DNA binding repeat units and an LHE variant designed or reprogrammed from an LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMTV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I- GpeMI, I-Gpil, I-GzeMI, I-GzeMII, I-GzeMin, I-HjeMI, I-Ltrll, I-Ltrl, I-LtrWI, I-MpeMI, I- MveMI, I-Ncrll, I-Ncrl, I-NcrMI, I-OheMI, I-Onul, I-OsoMI, I-OsoMII, I-OsoMIII, I- OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, I-Vdi 1411 and variants thereof, or preferably I-CpaMI, I-HjeMI, I-Onul, I-PanMI, SmaMI and variants thereof, or more preferably I-Onul and variants thereof.
In particular embodiments, a megaTAL contemplated herein, comprises an NTD, one or more TALE DNA binding repeat units, a CTD, and an LHE variant selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, I-ApaMI, I-CapHI, I-CapIV, I-CkaMI, I-CpaMI, I- CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-Gpil, I- GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I- Ncrl, I-NcrMI, I-OheMI, I-Onul, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I- PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, I-Vdil41I and variants thereof, or preferably I-CpaMI, I-HjeMI, I-Onul, I-PanMI, SmaMI and variants thereof, or more preferably I-Onul and variants thereof.
In particular embodiments, a megaTAL contemplated herein, comprises an NTD, about 9.5 to about 15.5 TALE DNA binding repeat units, and an LHE variant selected from the group consisting of: I-AabMI, I-AaeMI, I- Anil, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I- CpaMI, I-CpaMII, I-CpaMHI, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-Ej eMI, I-GpeMI, I- Gpil, I-GzeMI, I-GzeMII, I-GzeMHI, I-HjeMI, I-LtrII, I-Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I- Ncrll, I-Ncrl, I-NcrMI, I-OheMI, I-Onul, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, I-Vdil41I and variants thereof, or preferably I-CpaMI, I-HjeMI, I-Onul, I-PanMI, SmaMI and variants thereof, or more preferably I-Onul and variants thereof.
In particular embodiments, a megaTAL contemplated herein, comprises an NTD of about 122 amino acids to 137 amino acids, about 9.5, about 10.5, about 11.5, about 12.5, about 13.5, about 14.5, or about 15.5 binding repeat units, a CTD of about 20 amino acids to about 85 amino acids, and an I-Onul LHE variant. In particular embodiments, any one of, two of, or all of the NTD, DNA binding domain, and CTD can be designed from the same species or different species, in any suitable combination.
In particular embodiments, a megaTAL contemplated herein, comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-11.
In particular embodiments, a megaTAL-Trex2 fusion protein contemplated herein, comprises the amino acid sequence set forth in SEQ ID NO: 12.
In certain embodiments, a megaTAL comprises a TALE DNA binding domain and an I-Onul LHE variant binds and cleaves the nucleotide sequence set forth in SEQ ID NO: 15.
3. END-PROCESSING ENZYMES
Genome editing compositions and methods contemplated in particular embodiments comprise editing cellular genomes using a nuclease variant and an end-processing enzyme. In particular embodiments, a single polynucleotide encodes a homing endonuclease variant and an end-processing enzyme, separated by a linker, a self cleaving peptide sequence, e.g., 2A sequence, or by an IRES sequence. In particular embodiments, genome editing compositions comprise a polynucleotide encoding a nuclease variant and a separate polynucleotide encoding an end-processing enzyme.
The term "end-processing enzyme" refers to an enzyme that modifies the exposed ends of a polynucleotide chain. The polynucleotide may be double-stranded DNA (dsDNA), single- stranded DNA (ssDNA), RNA, double-stranded hybrids of DNA and RNA, and synthetic DNA (for example, containing bases other than A, C, G, and T). An end-processing enzyme may modify exposed polynucleotide chain ends by adding one or more nucleotides, removing one or more nucleotides, removing or modifying a phosphate group and/or removing or modifying a hydroxyl group. An end-processing enzyme may modify ends at endonuclease cut sites or at ends generated by other chemical or mechanical means, such as shearing (for example by passing through fine-gauge needle, heating, sonicating, mini bead tumbling, and nebulizing), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolysis and chemotherapy agents.
In particular embodiments, genome editing compositions and methods contemplated in particular embodiments comprise editing cellular genomes using a homing endonuclease variant or megaTAL and a DNA end-processing enzyme.
The term "DNA end-processing enzyme" refers to an enzyme that modifies the exposed ends of DNA. A DNA end-processing enzyme may modify blunt ends or staggered ends (ends with 5' or 3' overhangs). A DNA end-processing enzyme may modify single stranded or double stranded DNA. A DNA end-processing enzyme may modify ends at endonuclease cut sites or at ends generated by other chemical or mechanical means, such as shearing (for example by passing through fine-gauge needle, heating, sonicating, mini bead tumbling, and nebulizing), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolysis and chemotherapy agents. DNA end-processing enzyme may modify exposed DNA ends by adding one or more nucleotides, removing one or more nucleotides, removing or modifying a phosphate group and/or removing or modifying a hydroxyl group. Illustrative examples of DNA end-processing enzymes suitable for use in particular embodiments contemplated herein include, but are not limited to: 5 '-3' exonucleases, 5 '-3' alkaline exonucleases, 3 '-5' exonucleases, 5' flap endonucleases, helicases, phosphatases, hydrolases and template-independent DNA polymerases.
Additional illustrative examples of DNA end-processing enzymes suitable for use in particular embodiments contemplated herein include, but are not limited to, Trex2, Trexl, Trexl without transmembrane domain, Apollo, Artemis, DNA2, Exol, ExoT, ΕχοΠΙ, Fenl, Fanl, Mrell, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, Lambda exonuclease, Sox, Vaccinia DNA polymerase, exonuclease I, exonuclease III, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7-exonuclease Gene 6, avian myeloblastosis virus integration protein (IN), Bloom, Antartic Phophatase, Alkaline
Phosphatase, Poly nucleotide Kinase (PNK), Apel, Mung Bean nuclease, Hexl, TTRAP (TDP2), Sgsl, Sae2, CUP, Pol mu, Pol lambda, MUS81, EMEl, EME2, SLXl, SLX4 and UL- 12.
In particular embodiments, genome editing compositions and methods for editing cellular genomes contemplated herein comprise polypeptides comprising a homing
endonuclease variant or megaTAL and an exonuclease. The term "exonuclease" refers to enzymes that cleave phosphodiester bonds at the end of a polynucleotide chain via a hydrolyzing reaction that breaks phosphodiester bonds at either the 3 ' or 5 ' end.
Illustrative examples of exonucleases suitable for use in particular embodiments contemplated herein include, but are not limited to: hExoI, Yeast Exol, E. coli Exol, hTREX2, mouse TREX2, rat TREX2, hTREXl, mouse TREXl, rat TREX1, and Rat TREXl.
In particular embodiments, the DNA end-processing enzyme is a 3' or 5' exonuclease, preferably Trex 1 or Trex2, more preferably Trex2, and even more preferably human or mouse Trex2.
D. TARGET SITES
Nuclease variants contemplated in particular embodiments can be designed to bind to any suitable target sequence and can have a novel binding specificity, compared to a naturally- occurring nuclease. In particular embodiments, the target site is a regulatory region of a gene including, but not limited to promoters, enhancers, repressor elements, and the like. In particular embodiments, the target site is a coding region of a gene or a splice site. In certain embodiments, nuclease variants are designed to down-regulate or decrease expression of a gene. In particular embodiments, a nuclease variant and donor repair template can be designed to repair or delete a desired target sequence.
In various embodiments, nuclease variants bind to and cleave a target sequence in an interleukin 10 receptor 1 alpha (IL-lORa) gene. IL-lORa is also referred to as CDW210A, IL-10R1, IL-10RA, CD210 Antigen, ML-IOR, CD210a, CD210, and IBD28. The IL-lORa gene encodes a ~63kD protein that is expressed in the spleen, thymus, and PBMCs and is highly expressed in monocytes, B cells, large granular lymphocytes, and T cells. IL-lORa is also weakly expressed in pancreas, skeletal muscle, brain, heart, and kidney tissues, and intermediately expressed in placenta, lung, and liver tissue. IL-10 activates downstream signaling by binding to the IL-10 receptor (IL-IOR), comprised of two a subunits (encoded by IL-lORa) and two β subunits (encoded by IL-IORP). IL-10 mediates immunosuppressive signals via the IL- 1 ORa by inhibiting proinflammatory cytokine synthesis. Loss of IL- 1 ORa expression in regulatory T cells (Tregs) impairs the immune system's response to GVHD, inflammatory and autoimmune diseases.
In particular embodiments, a homing endonuclease variant or megaTAL introduces a double-strand break (DSB) in a target site in an IL-lORa gene. In particular
embodiments, a homing endonuclease variant or megaTAL introduces a DSB in exon 2 of an IL-lORa gene, preferably at SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of an IL- lORa gene, and more preferably at the sequence "ATTC" in SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of an IL-lORa gene.
In a preferred embodiment, a homing endonuclease variant or megaTAL cleaves double-stranded DNA and introduces a DSB into the polynucleotide sequence set forth in SEQ ID NO: 13 or 15.
In a preferred embodiment, the IL-lORa gene is a human IL-lORa gene. E. DONOR REPAIR TEMPLATES
Nuclease variants may be used to introduce a DSB in a target sequence; the DSB may be repaired through homology directed repair (HDR) mechanisms in the presence of one or more donor repair templates. In particular embodiments, the donor repair template is used to insert a sequence into the genome. In particular preferred embodiments, the donor repair template is used to repair or modify a sequence in the genome.
In various embodiments, a donor repair template is introduced into a hematopoietic cell, e.g., a T cell, by transducing the cell with an adeno-associated virus (AAV), retrovirus, e.g., lentivirus, IDLV, etc., herpes simplex virus, adenovirus, or vaccinia virus vector comprising the donor repair template.
In particular embodiments, the donor repair template comprises one or more homology arms that flank the DSB site.
As used herein, the term "homology arms" refers to a nucleic acid sequence in a donor repair template that is identical, or nearly identical, to DNA sequence flanking the DNA break introduced by the nuclease at a target site. In one embodiment, the donor repair template comprises a 5' homology arm that comprises a nucleic acid sequence that is identical or nearly identical to the DNA sequence 5 ' of the DNA break site. In one embodiment, the donor repair template comprises a 3' homology arm that comprises a nucleic acid sequence that is identical or nearly identical to the DNA sequence 3 ' of the DNA break site. In a preferred embodiment, the donor repair template comprises a 5 ' homology arm and a 3 ' homology arm. The donor repair template may comprise homology to the genome sequence immediately adjacent to the DSB site, or homology to the genomic sequence within any number of base pairs from the DSB site. In one embodiment, the donor repair template comprises a nucleic acid sequence that is homologous to a genomic sequence about 5 bp, about 10 bp, about 25 bp, about 50 bp, about 100 bp, about 250 bp, about 500 bp, about 1000 bp, about 2500 bp, about 5000 bp, about 10000 bp or more, including any intervening length of homologous sequence.
Illustrative examples of suitable lengths of homology arms contemplated in particular embodiments, may be independently selected, and include but are not limited to: about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, about 1500 bp, about 1600 bp, about 1700 bp, about 1800 bp, about 1900 bp, about 2000 bp, about 2100 bp, about 2200 bp, about 2300 bp, about 2400 bp, about 2500 bp, about 2600 bp, about 2700 bp, about 2800 bp, about 2900 bp, or about 3000 bp, or longer homology arms, including all intervening lengths of homology arms.
Additional illustrative examples of suitable homology arm lengths include, but are not limited to: about 100 bp to about 3000 bp, about 200 bp to about 3000 bp, about 300 bp to about 3000 bp, about 400 bp to about 3000 bp, about 500 bp to about 3000 bp, about 500 bp to about 2500 bp, about 500 bp to about 2000 bp, about 750 bp to about 2000 bp, about 750 bp to about 1500 bp, or about 1000 bp to about 1500 bp, including all intervening lengths of homology arms.
In a particular embodiment, the lengths of the 5' and 3 ' homology arms are independently selected from about 500 bp to about 1500 bp. In one embodiment, the
5'homology arm is about 1500 bp and the 3 ' homology arm is about 1000 bp. In one embodiment, the 5'homology arm is between about 200 bp to about 600 bp and the 3 ' homology arm is between about 200 bp to about 600 bp. In one embodiment, the 5 'homology arm is about 200 bp and the 3 ' homology arm is about 200 bp. In one embodiment, the 5'homology arm is about 300 bp and the 3 ' homology arm is about 300 bp. In one
embodiment, the 5'homology arm is about 400 bp and the 3 ' homology arm is about 400 bp. In one embodiment, the 5'homology arm is about 500 bp and the 3 ' homology arm is about 500 bp. In one embodiment, the 5'homology arm is about 600 bp and the 3 ' homology arm is about 600 bp.
Donor repair templates may further comprises one or more polynucleotides such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Art sites), termination codons, transcriptional termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, contemplated elsewhere herein.
In various embodiments, the donor repair template comprises a 5' homology arm, an RNA polymerase II promoter, one or more polynucleotides encoding a therapeutic gene or fragment thereof, transgene or selectable marker, and a 3 ' homology arm. In various embodiments, a target site is modified with a donor repair template comprising a 5' homology arm, one or more polynucleotides encoding self-cleaving viral peptide, e.g., T2A, a therapeutic gene or fragment thereof, transgene or selectable marker, optionally a poly(A) signal, and a 3 ' homology arm.
In various embodiments, the donor repair template comprises one or more
polynucleotides encoding a therapeutic gene or fragment thereof, transgene, or selectable marker.
In various embodiments, the donor repair template comprises one or more
polynucleotides encoding a gene or transgene selected from the group consisting of: a bispecific T cell engager (BiTE) molecule; a cytokine (e.g., IL-2, insulin, IFN-γ, IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-a), a chemokine (e.g., ΜΙΡ-Ια, ΜΙΡ-Ιβ, MCP-1, MCP-3, and RANTES), a cytotoxin (e.g., Perforin, Granzyme A, and Granzyme B), a cytokine receptor (e.g., an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, and an IL-21 receptor), and an engineered antigen receptor (e.g., an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a Daric receptor or components thereof, or a chimeric cytokine receptor receptor).
As used herein, the term "engineered TCR" refers to a T cell receptor, e.g., an αβ TCR that has a high-avidity and reactivity toward a target antigen. The engineered TCR may be selected, cloned, and subsequently introduced into a population of T cells used for adoptive immunotherapy. An engineered TCR is an exogenous TCR because it is introduced into T cells that do not normally express the particular TCR. The essential aspect of the engineered TCRs is that it has high avidity for a tumor antigen presented by a major histocompatibility complex (MHC) or similar immunological component. In contrast to engineered TCRs, CARs are engineered to bind target antigens in an MHC independent manner.
As used herein, the term "CAR" refers to a chimeric antigen receptor. Illustrative examples of CARs are disclosed in PCT Publication Nos. : WO2015164759, WO2015188119, and WO2016014789, each of which is incorporated herein by reference in its entirety.
As used herein, the term "Daric receptor" refers to a multichain engineered antigen receptor. Illustrative examples of Daric architectures and components thereof are disclosed in PCT Publication No. WO2015/017214 and U.S. Patent Publication No. 20150266973, each of which is incorporated herein by reference in its entirety.
As used herein, the terms "chimeric cytokine receptor" or "zetakine" refer to chimeric transmembrane immunoreceptors that comprise an extracellular domain comprising a soluble receptor ligand linked to a support region capable of tethering the extracellular domain to a cell surface, a transmembrane region and an intracellular signaling domain. Illustrative examples of zetakines are disclosed in U.S. Patent Nos.: 7,514,537; 8,324,353; 8,497,118; and
9,217,025, each of which is incorporated herein by reference in its entirety.
In one embodiment, the donor repair template comprises a polynucleotide comprising an IL-lORa gene or portion thereof and is designed to introduce one or more mutations in a genomic IL-lORa sequence such that a mutant IL-lORa gene product is expressed.
In one embodiment, the donor repair template comprises a polynucleotide encoding FoxP3, a polypeptide that increases expression of FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
In one embodiment, the donor repair template comprises a polynucleotide comprising an IL-lORa gene or portion thereof and is designed to correct one or more mutations in a genomic IL-lORa sequence such that a wild type IL-lORa gene product is expressed.
In one preferred embodiment, the donor template is designed such that a polynucleotide is inserted at a target site in the IL-lORa gene without substantially disrupting IL-lORa expression.
F. POLYPEPTIDES
Various polypeptides are contemplated herein, including, but not limited to, homing endonuclease variants, megaTALs, and fusion polypeptides. In preferred embodiments, a polypeptide comprises the amino acid sequence set forth in SEQ ID NOs: 1-12 and 21.
"Polypeptide," "polypeptide fragment," "peptide" and "protein" are used interchangeably, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids. In one embodiment, a "polypeptide" includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of well-known recombinant and/or synthetic techniques. Polypeptides are not limited to a specific length, e.g., they may comprise a full length protein sequence, a fragment of a full length protein, or a fusion protein, and may include post-translational modifications of the polypeptide, for example,
glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
An "isolated protein," "isolated peptide," or "isolated polypeptide" and the like, as used herein, refer to in vitro synthesis, isolation, and/or purification of a peptide or polypeptide molecule from a cellular environment, and from association with other components of the cell, i.e., it is not significantly associated with in vivo substances.
Illustrative examples of polypeptides contemplated in particular embodiments include, but are not limited to homing endonuclease variants, megaTALs, end-processing nucleases, fusion polypeptides and variants thereof.
Polypeptides include "polypeptide variants." Polypeptide variants may differ from a naturally occurring polypeptide in one or more amino acid substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more amino acids of the above polypeptide sequences. For example, in particular embodiments, it may be desirable to improve the biological properties of a homing endonuclease, megaTAL or the like that binds and cleaves a target site in the human IL-lORa gene by introducing one or more substitutions, deletions, additions and/or insertions into the polypeptide. In particular embodiments, polypeptides include polypeptides having at least about 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) amino acid identity to any of the reference sequences contemplated herein, typically where the variant maintains at least one biological activity of the reference sequence.
Polypeptides variants include biologically active "polypeptide fragments." Illustrative examples of biologically active polypeptide fragments include DNA binding domains, nuclease domains, and the like. As used herein, the term "biologically active fragment" or "minimal biologically active fragment" refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%), at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity. In preferred embodiments, the biological activity is binding affinity and/or cleavage activity for a target sequence. In certain embodiments, a polypeptide fragment can comprise an amino acid chain at least 5 to about 1700 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids long. In particular embodiments, a polypeptide comprises a biologically active fragment of a homing endonuclease variant. In particular embodiments, the polypeptides set forth herein may comprise one or more amino acids denoted as "X." "X" if present in an amino acid SEQ ID NO, refers to any amino acid. One or more "X" residues may be present at the N- and C-terminus of an amino acid sequence set forth in particular SEQ ID NOs contemplated herein. If the "X' amino acids are not present the remaining amino acid sequence set forth in a SEQ ID NO may be considered a biologically active fragment.
In particular embodiments, a polypeptide comprises a biologically active fragment of a homing endonuclease variant, e.g., SEQ ID NOs: 3-5. The biologically active fragment may comprise an N-terminal truncation and/or C-terminal truncation. In a particular embodiment, a biologically active fragment lacks or comprises a deletion of the 1, 2, 3, 4, 5, 6, 7, or 8 N- terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence, more preferably a deletion of the 4 N-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence. In a particular embodiment, a biologically active fragment lacks or comprises a deletion of the 1, 2, 3, 4, or 5 C-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence, more preferably a deletion of the 2 C-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence. In a particular preferred
embodiment, a biologically active fragment lacks or comprises a deletion of the 4 N-terminal amino acids and 2 C-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence. In a particular embodiment, an I-Onul variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or a deletion of the following 1, 2, 3, 4, or 5 C-terminal amino acids: R, G, S, F, V.
In a particular embodiment, an I-Onul variant comprises a deletion or substitution of 1, 2, 3, 4, 5, 6, 7, or 8 the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or a deletion or substitution of the following 1, 2, 3, 4, or 5 C-terminal amino acids: R, G, S, F, V.
In a particular embodiment, an I-Onul variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or a deletion of the following 1 or 2 C-terminal amino acids: F, V.
In a particular embodiment, an I-Onul variant comprises a deletion or substitution of 1,
2, 3, 4, 5, 6, 7, or 8 the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or a deletion or substitution of the following 1 or 2 C-terminal amino acids: F, V.
As noted above, polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367- 382), U.S. Pat. No. 4,873,192, Watson, J. D. et al, (Molecular Biology of the Gene, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif, 1987) and the references cited therein.
Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al, (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).
In certain embodiments, a variant will contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications may be made in the structure of the polynucleotides and polypeptides contemplated in particular embodiments, polypeptides include polypeptides having at least about and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant polypeptide, one skilled in the art, for example, can change one or more of the codons of the encoding DNA sequence, e.g., according to Table 1.
TABLE 1- Amino Acid Codons
Figure imgf000054_0001
Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR, DNA Strider, Geneious, Mac Vector, or Vector NTI software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non- polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson etal. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p.224).
In one embodiment, where expression of two or more polypeptides is desired, the polynucleotide sequences encoding them can be separated by and IRES sequence as disclosed elsewhere herein.
Polypeptides contemplated in particular embodiments include fusion polypeptides. In particular embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to a polypeptide having at least two, three, four, five, six, seven, eight, nine, or ten polypeptide segments.
In another embodiment, two or more polypeptides can be expressed as a fusion protein that comprises one or more self-cleaving polypeptide sequences as disclosed elsewhere herein.
In one embodiment, a fusion protein contemplated herein comprises one or more DNA binding domains and one or more nucleases, and one or more linker and/or self-cleaving polypeptides.
In one embodiment, a fusion protein contemplated herein comprises nuclease variant; a linker or self-cleaving peptide; and an end-processing enzyme including but not limited to a 5'- 3' exonuclease, a 5 '-3' alkaline exonuclease, and a 3 '-5' exonuclease {e.g., Trex2).
Fusion polypeptides can comprise one or more polypeptide domains or segments including, but are not limited to signal peptides, cell permeable peptide domains (CPP), DNA binding domains, nuclease domains, etc., epitope tags {e.g., maltose binding protein ("MBP"), glutathione S transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptide linkers, and polypeptide cleavage signals. Fusion polypeptides are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N- terminus, or N-terminus to C-terminus. In particular embodiments, the polypeptides of the fusion protein can be in any order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be produced by chemical synthetic methods or by chemical linkage between the two moieties or may generally be prepared using other standard techniques.
Ligated DNA sequences comprising the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as disclosed elsewhere herein.
Fusion polypeptides may optionally comprises a linker that can be used to link the one or more polypeptides or domains within a polypeptide. A peptide linker sequence may be employed to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structures so as to allow the polypeptide domains to exert their desired functions. Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al. , Gene 40:39-46, 1985; Murphy et al, Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. Linker sequences are not required when a particular fusion polypeptide segment contains non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference. Preferred linkers are typically flexible amino acid subsequences which are synthesized as part of a recombinant fusion protein. Linker polypeptides can be between 1 and 200 amino acids in length, between 1 and 100 amino acids in length, or between 1 and 50 amino acids in length, including all integer values in between.
Exemplary linkers include, but are not limited to the following amino acid sequences: glycine polymers (G)n; glycine-serine polymers (Gi-5Si-5)n, where n is an integer of at least one, two, three, four, or five; glycine-alanine polymers; alanine-serine polymers; GGG (SEQ ID NO: 23); DGGGS (SEQ ID NO: 24); TGEKP (SEQ ID NO: 25) (see e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 26) (Pomerantz etal. 1995, supra); (GGGGS)n wherein n = 1, 2, 3, 4 or 5 (SEQ ID NO: 27) (Kim etal, PNAS 93, 1156-1160 (1996.);
EGKSSGSGSESKVD (SEQ ID NO: 28) (Chaudhary et al. , 1990, Proc. Natl. Acad. Sci. U.S.A. 87: 1066-1070); KESGS VS SEQL AQFRSLD (SEQ ID NO: 29) (Bird et al, 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 30); LRQRDGERP (SEQ ID NO: 31);
LRQKDGGGSERP (SEQ ID NO: 32); LRQKD(GGGS)2ERP (SEQ ID NO: 33).
Alternatively, flexible linkers can be rationally designed using a computer program capable of modeling both DNA-binding sites and the peptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91 : 11099-11103 (1994) or by phage display methods.
Fusion polypeptides may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein or between an endogenous open reading frame and a polypeptide encoded by a donor repair template. In addition, a polypeptide cleavage site can be put into any linker peptide sequence. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).
Suitable protease cleavages sites and self-cleaving peptides are known to the skilled person (see, e.g., in Ryan et al, 1997. J Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include, but are not limited to the cleavage sites of poty virus NIa proteases (e.g., tobacco etch virus protease), poty virus HC proteases, poty virus PI (P35) proteases, byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus L proteases, enterovirus 2A proteases, rhinovirus 2A proteases, picorna 3C proteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungro spherical vims) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase. Due to its high cleavage stringency, TEV (tobacco etch virus) protease cleavage sites are preferred in one embodiment, e.g., EXXYXQ(G/S) (SEQ ID NO: 34), for example, ENLYFQG (SEQ ID NO: 35) and ENLYFQS (SEQ ID NO: 36), wherein X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S).
In certain embodiments, the self-cleaving polypeptide site comprises a 2A or 2A-like site, sequence or domain (Donnelly et al., 2001. J. Gen. Virol. 82: 1027-1041). In a particular embodiment, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.
In one embodiment, the viral 2A peptide is selected from the group consisting of: a foot-and-mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a Thosea asigna virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.
Illustrative examples of 2A sites are provided in Table 2.
TABLE 2: Exemplary 2A sites include the following sequences:
Figure imgf000058_0001
SEQ ID NO: 51 LLKLAGDVESNPGP
SEQ ID NO: 52 NFDLLKLAGDVESNPGP
SEQ ID NO: 53 QLLNFDLLKLAGDVESNPGP
SEQ ID NO: 54 APVKQTLNFDLLKLAGDVESNPGP
SEQ ID NO: 55 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT
SEQ ID NO: 56 LNFDLLKLAGDVESNPGP
SEQ ID NO: 57 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP
SEQ ID NO: 58 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP
G. POLYNUCLEOTIDES
In particular embodiments, polynucleotides encoding one or more homing
endonuclease variants, megaTALs, end-processing enzymes, and fusion polypeptides contemplated herein are provided. As used herein, the terms "polynucleotide" or "nucleic acid" refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA/RNA hybrids. Polynucleotides may be single-stranded or double-stranded and either recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA
(shRNA), microRNA (miRNA), ribozymes, genomic RNA (gRNA), plus strand RNA
(RNA(+)), minus strand RNA (RNA(-)), tracrRNA, crRNA, single guide RNA (sgRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA,
complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotides refer to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, as well as all intermediate lengths. It will be readily understood that "intermediate lengths, " in this context, means any length between the quoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc. ; 151, 152, 153, etc.; 201, 202, 203, etc. In particular embodiments, polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.
In particular embodiments, polynucleotides may be codon-optimized. As used herein, the term "codon-optimized" refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and/or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, (x) systematic variation of codon sets for each amino acid, and/or (xi) isolated removal of spurious translation initiation sites.
As used herein the term "nucleotide" refers to a heterocyclic nitrogenous base in N- glycosidic linkage with a phosphorylated sugar. Nucleotides are understood to include natural bases, and a wide variety of art-recognized modified bases. Such bases are generally located at the 1 ' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. In ribonucleic acid (RNA), the sugar is a ribose, and in deoxyribonucleic acid (DNA) the sugar is a deoxyribose, i.e., a sugar lacking a hydroxyl group that is present in ribose. Exemplary natural nitrogenous bases include the purines, adenosine (A) and guanidine (G), and the pyrimidines, cytidine (C) and thymidine (T) (or in the context of RNA, uracil (U)). The C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine. Nucleotides are usually mono, di- or triphosphates. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, nucleotide derivatives, modified nucleotides, non-natural nucleotides, and non-standard nucleotides; see for example, WO 92/07065 and WO 93/15187). Examples of modified nucleic acid bases are summarized by Limbach et al, (1994, Nucleic Acids Res. 22, 2183- 2196).
A nucleotide may also be regarded as a phosphate ester of a nucleoside, with esterification occurring on the hydroxyl group attached to C-5 of the sugar. As used herein, the term "nucleoside" refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases, and also to include well known modified bases. Such bases are generally located at the 1 ' position of a nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group. The nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides, or non-standard nucleosides). As also noted above, examples of modified nucleic acid bases are summarized by Limbach et al., (1994, Nucleic Acids Res. 22, 2183-2196).
Illustrative examples of polynucleotides include, but are not limited to polynucleotides encoding SEQ ID NOs: 1-12 and 21 and polynucleotide sequences set forth in SEQ ID NOs:
13-20.
In various illustrative embodiments, polynucleotides contemplated herein include, but are not limited to polynucleotides encoding homing endonuclease variants, megaTALs, end- processing enzymes, fusion polypeptides, and expression vectors, viral vectors, and transfer plasmids comprising polynucleotides contemplated herein.
As used herein, the terms "polynucleotide variant" and "variant" and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Accordingly, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides have been added or deleted, or modified, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide. In one embodiment, a polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions. To hybridize under "stringent conditions" describes hybridization protocols in which nucleotide sequences at least 60% identical to each other remain hybridized. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
The recitations "sequence identity" or, for example, comprising a "sequence 50% identical to," as used herein, refer to the extent that sequences are identical on a nucleotide-by- nucleotide basis or an amino acid-by -amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Tip, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.
Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al, Current Protocols in
Molecular Biology, John Wiley & Sons Inc., 1994-1998, Chapter 15.
An "isolated polynucleotide," as used herein, refers to a polynucleotide that has been purified from the sequences which flank it in a naturally -occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment. In particular embodiments, an "isolated polynucleotide" refers to a complementary DNA
(cDNA), a recombinant polynucleotide, a synthetic polynucleotide, or other polynucleotide that does not exist in nature and that has been made by the hand of man.
In various embodiments, a polynucleotide comprises an mRNA encoding a polypeptide contemplated herein including, but not limited to, a homing endonuclease variant, a megaTAL, and an end-processing enzyme. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.
As used herein, the terms "5' cap" or "5' cap structure" or "5' cap moiety" refer to a chemical modification, which has been incorporated at the 5' end of an mRNA. The 5' cap is involved in nuclear export, mRNA stability, and translation. In particular embodiments, a mRNA contemplated herein comprises a 5' cap comprising a 5 '-ppp-5 '-triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue.
Illustrative examples of 5' cap suitable for use in particular embodiments of the mRNA polynucleotides contemplated herein include, but are not limited to: unmethylated 5' cap analogs, e.g., G(5')ppp(5')G, G(5')ppp(5')C, G(5')ppp(5')A; methylated 5' cap analogs, e.g., m7G(5')ppp(5')G, m7G(5')ppp(5')C, and m7G(5')ppp(5')A; dimethylated 5' cap analogs, e.g., m2'7G(5')ppp(5')G, m2,7G(5')ppp(5')C, and m2,7G(5')ppp(5')A; trimethylated 5' cap analogs, e.g., m2'2'7G(5 ')ppp(5 ')G, m2A7G(5 ')ppp(5 ')C, and m2'2'7G(5 ')ρρρ(5 ')A; dimethylated symmetrical 5' cap analogs, e.g., m7G(5 ')pppm7(5 ')G, m7G(5')pppm7(5')C, and
m7G(5')pppm7(5')A; and anti reverse 5' cap analogs, e.g, Anti -Reverse Cap Analog (ARC A) cap, designated 3 O-Me-m7G(5')ppp(5')G, 2'0-Me-m7G(5')ppp(5')G, 2'0-Me- m7G(5')ppp(5')C, 2'0-Me-m7G(5')ppp(5')A, m72'd(5')ppp(5')G, m72'd(5')ppp(5')C, m72'd(5')ppp(5')A, 3'0-Me-m7G(5')ppp(5')C, 3 '0-Me-m7G(5')ppp(5')A,
m73 'd(5 ')ppp(5 ')G, m73 'd(5 ')ppp(5 ')C, m73 'd(5 ')ρρρ(5 ')A and their tetraphosphate derivatives) (see, e.g., Jemielity et al, RNA, 9: 1108-1122 (2003)).
In particular embodiments, mRNAs comprise a 5' cap that is a 7-methyl guanylate ("m7G") linked via a triphosphate bridge to the 5 '-end of the first transcribed nucleotide, resulting in m7G(5 ')ρρρ(5 ')N, where N is any nucleoside.
In some embodiments, mRNAs comprise a 5' cap wherein the cap is a CapO structure (CapO structures lack a 2'-0-methyl residue of the ribose attached to bases 1 and 2), a Capl structure (Capl structures have a 2'-0-methyl residue at base 2), or a Cap2 structure (Cap2 structures have a 2'-0-methyl residue attached to both bases 2 and 3).
In one embodiment, an mRNA comprises a m7G(5 )ppp(5 ')G cap.
In one embodiment, an mRNA comprises an ARCA cap.
In particular embodiments, an mRNA contemplated herein comprises one or more modified nucleosides.
In one embodiment, an mRNA comprises one or more modified nucleosides selected from the group consisting of: pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2- thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl -uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl- uridine, 1 -methyl -pseudouri dine, 4-thio- 1 -methyl -pseudouri dine, 2-thio-l-methyl- pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydroundine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy -4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy -2-thio- pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1 -methyl -pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1 -methyl -pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocyti dine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy- 5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1 -methyl -pseudoisocytidine, 2- aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2- aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2- methoxy-adenine, inosine, 1 -methyl -inosine, wyosine, wybutosine, 7-deaza-guanosine, 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy- guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo- guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio- guanosine, and N2,N2-dimethyl-6-thio-guanosine.
In one embodiment, an mRNA comprises one or more modified nucleosides selected from the group consisting of: pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2- thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl -uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl- uridine, 1 -methyl -pseudouri dine, 4-thio-l -methyl -pseudouri dine, 2-thio-l-methyl- pseudouri dine, 1 -methyl- 1-deaza-pseudouri dine, 2-thio-l -methyl- 1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio- pseudouridine.
In one embodiment, an mRNA comprises one or more modified nucleosides selected from the group consisting of: 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4- acetylcytidine, 5-formyl cyti dine, N4-methylcyti dine, 5-hydroxymethylcytidine, 1 -methyl - pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l -methyl -pseudoisocytidine, 4-thio-l - methyl-l-deaza-pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocyti dine, zebularine, 5-aza- zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l- methyl-pseudoi socyti dine .
In one embodiment, an mRNA comprises one or more modified nucleosides selected from the group consisting of: 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8- aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.
In one embodiment, an mRNA comprises one or more modified nucleosides selected from the group consisting of: inosine, 1 -methyl -inosine, wyosine, wybutosine, 7-deaza- guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7- deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6- methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8- oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio- guanosine, and N2,N2-dimethyl-6-thio-guanosine.
In one embodiment, an mRNA comprises one or more pseudouridines, one or more 5- methyl-cytosines, and/or one or more 5-methyl-cytidines.
In one embodiment, an mRNA comprises one or more pseudouridines.
In one embodiment, an mRNA comprises one or more 5-methyl-cytidines.
In one embodiment, an mRNA comprises one or more 5-methyl-cytosines.
In particular embodiments, an mRNA contemplated herein comprises a poly(A) tail to help protect the mRNA from exonuclease degradation, stabilize the mRNA, and facilitate translation. In certain embodiments, an mRNA comprises a 3' poly(A) tail structure.
In particular embodiments, the length of the poly(A) tail is at least about 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or at least about 500 or more adenine nucleotides or any intervening number of adenine nucleotides. In particular embodiments, the length of the poly(A) tail is at least about 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 202, 203, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, or 275 or more adenine nucleotides.
In particular embodiments, the length of the poly(A) tail is about 10 to about 500 adenine nucleotides, about 50 to about 500 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 250 to about 500 adenine nucleotides, about 300 to about 500 adenine nucleotides, about 50 to about 450 adenine nucleotides, about 50 to about 400 adenine nucleotides, about 50 to about 350 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 100 to about 450 adenine nucleotides, about 100 to about 400 adenine nucleotides, about 100 to about 350 adenine nucleotides, about 100 to about 300 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 150 to about 450 adenine nucleotides, about 150 to about 400 adenine nucleotides, about 150 to about 350 adenine nucleotides, about 150 to about 300 adenine nucleotides, about 150 to about 250 adenine nucleotides, about 150 to about 200 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 200 to about 450 adenine nucleotides, about 200 to about 400 adenine nucleotides, about 200 to about 350 adenine nucleotides, about 200 to about 300 adenine nucleotides, about 250 to about 500 adenine nucleotides, about 250 to about 450 adenine nucleotides, about 250 to about 400 adenine nucleotides, about 250 to about 350 adenine nucleotides, or about 250 to about 300 adenine nucleotides or any intervening range of adenine nucleotides.
Terms that describe the orientation of polynucleotides include: 5' (normally the end of the polynucleotide having a free phosphate group) and 3 ' (normally the end of the
polynucleotide having a free hydroxyl (OH) group). Polynucleotide sequences can be annotated in the 5 ' to 3 ' orientation or the 3 ' to 5 ' orientation. For DNA and mRNA, the 5 ' to 3 ' strand is designated the "sense," "plus," or "coding" strand because its sequence is identical to the sequence of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA, instead of thymine (T) in DNA]. For DNA and mRNA, the complementary 3 ' to 5' strand which is the strand transcribed by the RNA polymerase is designated as "template," "antisense," "minus," or "non-coding" strand. As used herein, the term "reverse orientation" refers to a 5' to 3' sequence written in the 3 ' to 5 ' orientation or a 3 ' to 5 ' sequence written in the 5 ' to 3 ' orientation.
The terms "complementary" and "complementarity" refer to polynucleotides {i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the complementary strand of the DNA sequence 5' A G T C A T G 3' is 3' T C A G T A C 5'. The latter sequence is often written as the reverse complement with the 5 ' end on the left and the 3 ' end on the right, 5 C A T G A C T 3'. A sequence that is equal to its reverse complement is said to be a palindromic sequence. Complementarity can be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there can be "complete" or "total" complementarity between the nucleic acids. The term "nucleic acid cassette" or "expression cassette" as used herein refers to genetic sequences within the vector which can express an RNA, and subsequently a polypeptide. In one embodiment, the nucleic acid cassette contains a gene(s)-of-interest, e.g., a polynucleotide(s)-of-interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, enhancer, poly(A) sequence, and a gene(s)-of-interest, e.g., a polynucleotide(s)-of-interest. Vectors may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleic acid cassettes. The nucleic acid cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or a polypeptide, undergo appropriate post-translational modifications required for activity in the transformed cell, and be translocated to the appropriate compartment for biological activity by targeting to appropriate intracellular compartments or secretion into extracellular compartments. Preferably, the cassette has its 3' and 5' ends adapted for ready insertion into a vector, e.g., it has restriction endonuclease sites at each end. In a preferred embodiment, the nucleic acid cassette contains the sequence of a therapeutic gene used to treat, prevent, or ameliorate a genetic disorder. The cassette can be removed and inserted into a plasmid or viral vector as a single unit.
Polynucleotides include polynucleotide(s)-of-interest. As used herein, the term "polynucleotide-of-interest" refers to a polynucleotide encoding a polypeptide or fusion polypeptide or a polynucleotide that serves as a template for the transcription of an inhibitory polynucleotide, as contemplated herein.
Moreover, it will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that may encode a polypeptide, or fragment of variant thereof, as contemplated herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene.
Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, for example polynucleotides that are optimized for human and/or primate codon selection. In one embodiment, polynucleotides comprising particular allelic sequences are provided. Alleles are endogenous polynucleotide sequences that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. In a certain embodiment, a polynucleotide-of-interest comprises a donor repair template.
In a certain embodiment, a polynucleotide-of-interest comprises an inhibitory polynucleotide including, but not limited to, an siRNA, an miRNA, an shRNA, a ribozyme or another inhibitory RNA.
In one embodiment, a donor repair template comprising an inhibitory RNA comprises one or more regulatory sequences, such as, for example, a strong constitutive pol ΠΙ, e.g., human or mouse U6 snRNA promoter, the human and mouse HI RNA promoter, or the human tRNA-val promoter, or a strong constitutive pol Π promoter, as described elsewhere herein.
The polynucleotides contemplated in particular embodiments, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Art sites), termination codons,
transcriptional termination signals, post-transcription response elements, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably. It is therefore contemplated in particular embodiments that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol .
Polynucleotides can be prepared, manipulated, expressed and/or delivered using any of a variety of well-established techniques known and available in the art. In order to express a desired polypeptide, a nucleotide sequence encoding the polypeptide, can be inserted into appropriate vector. A desired polypeptide can also be expressed by delivering an mRNA encoding the polypeptide into the cell.
Illustrative examples of vectors include, but are not limited to plasmid, autonomously replicating sequences, and transposable elements, e.g., Sleeping Beauty, PiggyBac.
Additional illustrative examples of vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses.
Illustrative examples of viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).
Illustrative examples of expression vectors include, but are not limited to pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST™, pLenti6/V5- DEST™, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, coding sequences of polypeptides disclosed herein can be ligated into such expression vectors for the expression of the polypeptides in mammalian cells.
In particular embodiments, the vector is an episomal vector or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that is able to replicate without integration into host's chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally.
"Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are those non-translated regions of the vector— origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, post-transcriptional regulatory elements, a
polyadenylation sequence, 5 ' and 3 ' untranslated regions— which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters may be used.
In particular embodiments, a polynucleotide comprises a vector, including but not limited to expression vectors and viral vectors. A vector may comprise one or more exogenous, endogenous, or heterologous control sequences such as promoters and/or enhancers. An "endogenous control sequence" is one which is naturally linked with a given gene in the genome. An "exogenous control sequence" is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter. A "heterologous control sequence" is an exogenous sequence that is from a different species than the cell being genetically manipulated. A "synthetic" control sequence may comprise elements of one more endogenous and/or exogenous sequences, and/or sequences determined in vitro or in silico that provide optimal promoter and/or enhancer activity for the particular therapy.
The term "promoter" as used herein refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter. In particular embodiments, promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.
The term "enhancer" refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and/or other enhancer elements. The term "promoter/enhancer" refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions.
The term "operably linked", refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and/or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide- of-interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter/enhancer that continually or continuously allows for transcription of an operably linked sequence. A constitutive expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter/enhancer that allows expression in a wide variety of cell and tissue types or a "cell specific," "cell type specific," "cell lineage specific," or "tissue specific" promoter, enhancer, or promoter/enhancer that allows expression in a restricted variety of cell and tissue types, respectively.
Illustrative ubiquitous expression control sequences suitable for use in particular embodiments include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and PI 1 promoters from vaccinia virus, a short elongation factor 1 -alpha (EF la-short) promoter, a long elongation factor 1 -alpha (EF la-long) promoter, early growth response 1 (EGRl), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β-ΚΠΝΓ), the human ROSA 26 locus (Mans et al, Nature Biotechnology 25, 1477 - 1482 (2007)), a Ubiquitin C promoter (UBC), a phosphogly cerate kinase- 1 (PGK) promoter, a cytomegalovirus enhancer/chicken β-actin (CAG) promoter, a β-actin promoter and a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter (Challita et al, J Virol. 69(2):748-55 (1995)).
In a particular embodiment, it may be desirable to use a cell, cell type, cell lineage or tissue specific expression control sequence to achieve cell type specific, lineage specific, or tissue specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues or during specific stages of development).
As used herein, "conditional expression" may refer to any type of conditional expression including, but not limited to, inducible expression; repressible expression;
expression in cells or tissues having a particular physiological, biological, or disease state, etc. This definition is not intended to exclude cell type or tissue specific expression. Certain embodiments provide conditional expression of a polynucleotide-of-interest, e.g., expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that causes an increase or decrease in expression of the polynucleotide encoded by the polynucleotide-of-interest. Illustrative examples of inducible promoters/sy stems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the "GeneSwitch" mifepristone-regulatable system (Sirin etal., 2003, Gene, 323:67), the cumate inducible gene switch (WO 2002/088346), tetracycline-dependent regulatory systems, etc.
Conditional expression can also be achieved by using a site specific DNA recombinase. According to certain embodiments, polynucleotides comprises at least one (typically two) site(s) for recombination mediated by a site specific recombinase. As used herein, the terms "recombinase" or "site specific recombinase" include excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, six, seven, eight, nine, ten or more.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in particular embodiments include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, OC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCEl, and ParA.
The polynucleotides may comprise one or more recombination sites for any of a wide variety of site specific recombinases. It is to be understood that the target site for a site specific recombinase is in addition to any site(s) required for integration of a vector, e.g., a retroviral vector or lentiviral vector. As used herein, the terms "recombination sequence,"
"recombination site," or "site specific recombination site" refer to a particular nucleic acid sequence to which a recombinase recognizes and binds.
For example, one recombination site for Cre recombinase is loxP which is a 34 base pair sequence comprising two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence (see FIG. 1 of Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Other exemplary loxP sites include, but are not limited to: lox511 (Hoess et al. , 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), 1οχ2272 (Lee and Saito, 1998), m2 (Langer et al, 2002), lox71 (Albert et al, 1995), and lox66 (Albert et al., 1995).
Suitable recognition sites for the FLP recombinase include, but are not limited to: FRT (McLeod, et al, 1996), Fi,F2, F3 (Schlake and Bode, 1994), F4,F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al, 1988), FRT(RE) (Senecoff et al, 1988).
Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme λ Integrase, e.g., phi-c31. The ^C31 SSR mediates recombination only between the heterotypic sites attB (34 bp in length) and attP (39 bp in length) (Groth et al, 2000). attB and attP, named for the attachment sites for the phage integrase on the bacterial and phage genomes, respectively, both contain imperfect inverted repeats that are likely bound by ^C31 homodimers (Groth et al, 2000). The product sites, attL and attR, are effectively inert to further φΟ> 1-mediated recombination (Belteki et al, 2003), making the reaction irreversible. For catalyzing insertions, it has been found that attB-bearing DNA inserts into a genomic attP site more readily than an attP site into a genomic attB site (Thyagaraj an et al. , 2001 ; Belteki et al. , 2003). Thus, typical strategies position by homologous recombination an attP-bearing "docking site" into a defined locus, which is then partnered with an attB-bearing incoming sequence for insertion.
In one embodiment, a polynucleotide contemplated herein comprises a donor repair template polynucleotide flanked by a pair of recombinase recognition sites. In particular embodiments, the repair template polynucleotide is flanked by LoxP sites, FRT sites, or art sites.
In particular embodiments, polynucleotides contemplated herein, include one or more polynucleotides-of-interest that encode one or more polypeptides. In particular embodiments, to achieve efficient translation of each of the plurality of polypeptides, the polynucleotide sequences can be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides.
As used herein, an "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene. See, e.g., Jackson et al, 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES generally employed by those of skill in the art include those described in U.S. Pat. No. 6,692,736. Further examples of "IRES" known in the art include, but are not limited to IRES obtainable from picomavirus (Jackson et al, 1990) and IRES obtainable from viral or cellular mRNA sources, such as for example, immunoglobulin heavy-chain binding protein (BiP), the vascular endothelial growth factor (VEGF) (Huez et al. 1998. Mol. Cell. Biol. 18(11):6178-6190), the fibroblast growth factor 2 (FGF-2), and insulinlike growth factor (IGFII), the translational initiation factor eIF4G and yeast transcription factors TFIJD and HAP4, the encephelomycarditis virus (EMCV) which is commercially available from Novagen (Duke et al, 1992. J. Virol 66(3): 1602-9) and the VEGF IRES (Huez et al, 1998. Mol Cell Biol 18(11):6178-90). IRES have also been reported in viral genomes of Picornaviridae, Dicistroviridae and Flaviviridae species and in HCV, Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV).
In one embodiment, the IRES used in polynucleotides contemplated herein is an EMCV IRES.
In particular embodiments, the polynucleotides comprise polynucleotides that have a consensus Kozak sequence and that encode a desired polypeptide. As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA to the small subunit of the ribosome and increases translation. The consensus Kozak sequence is (GCC)RCCATGG [SEQ ID NO: 59], where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48).
Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increases heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. In particular embodiments, vectors comprise a polyadenylation sequence 3 Of a polynucleotide encoding a polypeptide to be expressed. The terms "polyA site," "polyA sequence," "poly(A) site" or "poly(A) sequence" as used herein denote a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a poly(A) tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly(A) tail are unstable and are rapidly degraded. Illustrative examples of poly (A) signals that can be used in a vector, includes an ideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA), a bovine growth hormone poly(A) sequence (BGHpA), a rabbit β-globin poly(A) sequence (rPgpA), or another suitable heterologous or endogenous poly(A) sequence known in the art.
In some embodiments, a polynucleotide or cell harboring the polynucleotide utilizes a suicide gene, including an inducible suicide gene to reduce the risk of direct toxicity and/or uncontrolled proliferation. In specific embodiments, the suicide gene is not immunogenic to the host harboring the polynucleotide or cell. A certain example of a suicide gene that may be used is caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).
In certain embodiments, polynucleotides comprise gene segments that cause the genetically modified cells contemplated herein to be susceptible to negative selection in vivo. "Negative selection" refers to an infused cell that can be eliminated as a result of a change in the in vivo condition of the individual. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selection genes are known in the art, and include, but are not limited to: the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyl transferase (APRT) gene, and bacterial cytosine deaminase.
In some embodiments, genetically modified cells comprise a polynucleotide further comprising a positive marker that enables the selection of cells of the negative selectable phenotype in vitro. The positive selectable marker may be a gene, which upon being introduced into the host cell, expresses a dominant phenotype permitting positive selection of cells carrying the gene. Genes of this type are known in the art, and include, but are not limited to hygromycin-B phosphotransferase gene (hph) which confers resistance to hygromycin B, the amino glycoside phosphotransferase gene (neo or aph) from Tn5 which codes for resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drug resistance (MDR) gene.
In one embodiment, the positive selectable marker and the negative selectable element are linked such that loss of the negative selectable element necessarily also is accompanied by loss of the positive selectable marker. In a particular embodiment, the positive and negative selectable markers are fused so that loss of one obligatorily leads to loss of the other. An example of a fused polynucleotide that yields as an expression product a polypeptide that confers both the desired positive and negative selection features described above is a hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene yields a polypeptide that confers hygromycin B resistance for positive selection in vitro, and ganciclovir sensitivity for negative selection in vivo. See also the publications of PCT US91/08442 and PCT/US94/05601, by S. D. Lupton, describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable markers with negative selectable markers.
Preferred positive selectable markers are derived from genes selected from the group consisting of hph, nco, and gpt, and preferred negative selectable markers are derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt. Exemplary bifunctional selectable fusion genes contemplated in particular embodiments include, but are not limited to genes wherein the positive selectable marker is derived from hph or neo, and the negative selectable marker is derived from cytosine deaminase or a TK gene or selectable marker.
In particular embodiments, polynucleotides encoding one or more nuclease variants, megaTALs, end-processing enzymes, or fusion polypeptides may be introduced into hematopoietic cells, e.g., T cells, by both non-viral and viral methods. In particular embodiments, delivery of one or more polynucleotides encoding nucleases and/or donor repair templates may be provided by the same method or by different methods, and/or by the same vector or by different vectors.
The term "vector" is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA. In particular embodiments, non-viral vectors are used to deliver one or more polynucleotides contemplated herein to a T cell. Illustrative examples of non-viral vectors include, but are not limited to plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.
Illustrative methods of non-viral delivery of polynucleotides contemplated in particular embodiments include, but are not limited to: electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene gun, and heat-shock.
Illustrative examples of polynucleotide delivery systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are sold commercially (e.g.,
Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides have been described in the literature. See e.g., Liu et al. (2003) Gene Therapy. 10: 180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011 : 1-12. Antibody -targeted, bacterially derived, non-living nanocell-based delivery is also contemplated in particular embodiments.
Viral vectors comprising polynucleotides contemplated in particular embodiments 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. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsy, etc.) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient.
In one embodiment, viral vectors comprising nuclease variants and/or donor repair templates are administered directly to an organism for transduction of cells in vivo.
Alternatively, naked DNA 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. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
Illustrative examples of viral vector systems suitable for use in particular
embodiments contemplated herein include, but are not limited to adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.
In various embodiments, one or more polynucleotides encoding a nuclease variant and/or donor repair template are introduced into a hematopoietic cell, e.g., a T cell, by transducing the cell with a recombinant adeno-associated virus (rAAV), comprising the one or more polynucleotides.
AAV is a small (-26 nm) replication-defective, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. Recombinant AAV (rAAV) are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3 ' AAV inverted terminal repeats (ITRs). The ITR sequences are about 145 bp in length. In particular embodiments, the rAAV comprises ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.
In some embodiments, a chimeric rAAV is used the ITR sequences are isolated from one AAV serotype and the capsid sequences are isolated from a different AAV serotype. For example, a rAAV with ITR sequences derived from AAV2 and capsid sequences derived from AAV6 is referred to as AAV2/AAV6. In particular embodiments, the rAAV vector may comprise ITRs from AAV2, and capsid proteins from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV6. In a preferred embodiment, the rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV2.
In some embodiments, engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest. Construction of rAAV vectors, production, and purification thereof have been disclosed, e.g., in U.S. Patent Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated by reference herein, in its entirety.
In various embodiments, one or more polynucleotides encoding a nuclease variant and/or donor repair template are introduced into a hematopoietic cell, by transducing the cell with a retrovirus, e.g., lentivirus, comprising the one or more polynucleotides.
As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Illustrative retroviruses suitable for use in particular embodiments, include, but are not limited to: Moloney murine leukemia virus (M- MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus
(HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentivirus.
As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human
immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV based vector backbones (i.e., HIV cis-acting sequence elements) are preferred.
In various embodiments, a lentiviral vector contemplated herein comprises one or more LTRs, and one or more, or all, of the following accessory elements: a cPPT/FLAP, a Psi (Ψ) packaging signal, an export element, poly (A) sequences, and may optionally comprise a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as discussed elsewhere herein.
In particular embodiments, lentiviral vectors contemplated herein may be integrative or non-integrating or integration defective lentivirus. As used herein, the term "integration defective lentivirus" or "IDLV" refers to a lentivirus having an integrase that lacks the capacity to integrate the viral genome into the genome of the host cells. Integration-incompetent viral vectors have been described in patent application WO 2006/010834, which is herein incorporated by reference in its entirety.
Illustrative mutations in the HIV-1 pol gene suitable to reduce integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H.
In one embodiment, the HIV-1 integrase deficient pol gene comprises a D64V, Dl 161,
Dl 16A, E152G, or E152A mutation; D64V, Dl 161, and E152G mutations; or D64V, Dl 16A, and El 52 A mutations.
In one embodiment, the HIV-1 integrase deficient pol gene comprises a D64V mutation.
The term "long terminal repeat (LTR)" refers to domains of base pairs located at the ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain U3, R and U5 regions.
As used herein, the term "FLAP element" or "cPPT/FLAP" refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, etal, 2000, Cell, 101 : 173. In another embodiment, a lentiviral vector contains a FLAP element with one or more mutations in the cPPT and/or CTS elements. In yet another embodiment, a lentiviral vector comprises either a cPPT or CTS element. In yet another embodiment, a lentiviral vector does not comprise a cPPT or CTS element.
As used herein, the term "packaging signal" or "packaging sequence" refers to psi [Ψ] sequences located within the retroviral genome which are required for insertion of the viral RNA into the viral capsid or particle, see e.g., Clever etal, 1995. J of Virology, Vol. 69, No. 4; pp. 2101-2109. The term "export element" refers to a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human
immunodeficiency virus (HIV) rev response element (RRE) (see e.g., Cullen et al., 1991. J Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), and the hepatitis B virus post- transcriptional regulatory element (HPRE).
In particular embodiments, expression of heterologous sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors. A variety of posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid at the protein, e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al. , 1999, J. Virol., 73 :2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al, Mol. Cell. Biol, 5:3864); and the like (Liu et al, 1995, Genes Dev., 9: 1766).
Lentiviral vectors preferably contain several safety enhancements as a result of modifying the LTRs. "Self-inactivating" (SIN) vectors refers to replication-defective vectors, e.g., in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. An additional safety enhancement is provided by replacing the U3 region of the 5 ' LTR with a heterologous promoter to drive transcription of the viral genome during production of viral particles. Examples of heterologous promoters which can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.
The terms "pseudotype" or "pseudotyping" as used herein, refer to a virus whose viral envelope proteins have been substituted with those of another virus possessing preferable characteristics. For example, HIV can be pseudotyped with vesicular stomatitis virus G-protein (VSV-G) envelope proteins, which allows HIV to infect a wider range of cells because HIV envelope proteins (encoded by the env gene) normally target the virus to CD4+ presenting cells. In certain embodiments, lentiviral vectors are produced according to known methods. See e.g., Kutner et al, BMC Biotechnol. 2009;9: 10. doi: 10.1186/1472-6750-9-10; Kutner et a/. Nat. Protoc. 2009;4(4):495-505. doi: 10.1038/nprot.2009.22.
According to certain specific embodiments contemplated herein, most or all of the viral vector backbone sequences are derived from a lentivirus, e.g., HIV-1. However, it is to be understood that many different sources of retroviral and/or lentiviral sequences can be used, or combined and numerous substitutions and alterations in certain of the lentiviral sequences may be accommodated without impairing the ability of a transfer vector to perform the functions described herein. Moreover, a variety of lentiviral vectors are known in the art, see Naldini et al, (1996a, 1996b, and 1998); Zufferey et al, (1997); Dull et al, 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which may be adapted to produce a viral vector or transfer plasmid contemplated herein.
In various embodiments, one or more polynucleotides encoding a nuclease variant and/or donor repair template are introduced into a hematopoietic cell by transducing the cell with an adenovirus comprising the one or more polynucleotides.
Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Most adenovirus vectors are engineered such that a transgene replaces the Ad El a, Elb, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity.
Generation and propagation of the current adenovirus vectors, which are replication deficient, may utilize a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones & Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham & Prevec, 1991 ).
Adenovirus vectors have been used in eukary otic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al, 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz & Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al, 1993). An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al, Hum. Gene Ther. 7: 1083-9 (1998)).
In various embodiments, one or more polynucleotides encoding nuclease variant and/or donor repair template are introduced into a hematopoietic cell by transducing the cell with a herpes simplex virus, e.g., HSV-1, HSV-2, comprising the one or more polynucleotides.
The mature HSV virion consists of an enveloped icosahedral capsid with a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb. In one embodiment, the HSV based viral vector is deficient in one or more essential or non-essential HSV genes. In one embodiment, the HSV based viral vector is replication deficient. Most replication deficient HSV vectors contain a deletion to remove one or more intermediate-early, early, or late HSV genes to prevent replication. For example, the HSV vector may be deficient in an immediate early gene selected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and a combination thereof. Advantages of the HSV vector are its ability to enter a latent stage that can result in long-term DNA expression and its large viral DNA genome that can accommodate exogenous DNA inserts of up to 25 kb. HSV-based vectors are described in, for example, U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583, each of which are incorporated by reference herein in its entirety.
H. GENOME EDITED CELLS
The genome edited cells manufactured by the methods contemplated in particular embodiments comprise one or more gene edits in an IL-lORa gene and provide improved cell- based therapeutics for the prevention, treatment, or amelioration of at least one symptom, of a cancer, GVHD, infectious disease, autoimmune disease, immunodeficiency or condition associated therewith. Without wishing to be bound to any particular theory, it is believed that the compositions and methods contemplated herein increase the efficacy of adoptive cell therapies, in part, by making the therapeutic cells more resistant to immunosuppressive signals and exhaustion. It is also believed that the compositions and methods contemplated herein restore the potential of immune cells to respond to inflammatory and autoimmune diseases.
Genome edited cells contemplated in particular embodiments may be
autologous/autogeneic ("self) or non-autologous ("non-self," e.g., allogeneic, syngeneic or xenogeneic). "Autologous," as used herein, refers to cells from the same subject.
"Allogeneic," as used herein, refers to cells of the same species that differ genetically to the cell in comparison. "Syngeneic," as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison. "Xenogeneic," as used herein, refers to cells of a different species to the cell in comparison. In preferred embodiments, the cells are obtained from a mammalian subject. In a more preferred embodiment, the cells are obtained from a primate subject, optionally a non-human primate. In the most preferred embodiment, the cells are obtained from a human subject.
An "isolated cell" refers to a non-naturally occurring cell, e.g., a cell that does not exist in nature, a modified cell, an engineered cell, etc., that has been obtained from an in vivo tissue or organ and is substantially free of extracellular matrix.
As used herein, the term "population of cells" refers to a plurality of cells that may be made up of any number and/or combination of homogenous or heterogeneous cell types, as described elsewhere herein. For example, for transduction of T cells, a population of cells may be isolated or obtained from peripheral blood. A population of cells may comprise about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%), or about 100%> of the target cell type to be edited. In certain embodiments, T cells may be isolated or purified from a population of heterogeneous cells using methods known in the art.
Illustrative examples of cell types whose genome can be edited using the compositions and methods contemplated herein include, but are not limited to, cell lines, primary cells, stem cells, progenitor cells, and differentiated cells, and mixtures thereof.
In a preferred embodiment, the genome editing compositions and methods are used to edit hematopoietic cells, more preferably immune cells, and even more preferably T cells. The terms "T cell" or "T lymphocyte" are art-recognized and are intended to include thymocytes, immune effector cells, regulatory T cells, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4+ T cell) CD4+ T cell, a cytotoxic T cell (CTL; CD8+ T cell), a tumor infiltrating cytotoxic T cell (TIL; CD8+ T cell), CD4+CD8+ T cell, CD4 CD8" T cell, or any other subset of T cells. In one embodiment, the T cell is an immune effector cell. In one embodiment, the T cell is a Treg. In one embodiment, the T cell is an NKT cell. Other illustrative populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells.
In various embodiments, genome edited cells comprise immune effector cells comprising an IL-lORa gene edited by the compositions and methods contemplated herein. An "immune effector cell," is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). Illustrative immune effector cells contemplated in particular embodiments are T lymphocytes, in particular cytotoxic T cells (CTLs; CD8+ T cells), TILs, and helper T cells (HTLs; CD4+ T cells). In one embodiment, immune effector cells include natural killer (NK) cells. In one embodiment, immune effector cells include natural killer T (NKT) cells.
In particular embodiments, T cells also include "regulatory T cells" or "Tregs." As used herein the terms "regulatory T cells" or "Tregs" are used interchangeably and refer to subsets of T cells that suppress immune and inflammatory responses to both self and foreign antigens. In particular embodiments, Tregs suppress the proliferation or cytokine production of activated T cells. In some embodiments, Tregs directly suppress autoantibody production of autoreactive B cells. In other embodiments, Tregs modulate an inflammatory response by regulating activation of myeloid and endothelial cells. Regulatory T cells are derived from the thymus (tTreg) or periphery (pTreg). Tregs may be derived from CD4+ cells (CD4+ Tregs) or CD8+ cells (CD8+ Tregs). Tregs express FoxP3 and cell surface markers including, but not limited, to CD4, CD25, GITR or CTLA4. pTreg and tTreg subsets can also be identified on the basis of Helios expression. Some regulatory T cell subsets, such as Trl cells, are FoxP3- deficient, and can be identified on the basis of CD49b and Lag3 expression. Tregs can mediate immunosuppressive activity through both contact dependent (e.g., Granzyme B) or contact independent processes (e.g., by producing immunosuppressive cytokines, including but not limited to, IL10, IL35 and TGFb l).
"Potent T cells," and "young T cells," are used interchangeably in particular embodiments and refer to T cell phenotypes wherein the T cell is capable of proliferation and a concomitant decrease in differentiation. In particular embodiments, the young T cell has the phenotype of a "naive T cell." In particular embodiments, young T cells comprise one or more of, or all of the following biological markers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, and CD38. In one embodiment, young T cells comprise one or more of, or all of the following biological markers: CD62L, CD127, CD197, and CD38. In one embodiment, the young T cells lack expression of CD57, CD244, CD160, PD-1, CTLA4, ΉΜ3, and LAG3.
T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
In particular embodiments, a population of cells comprising immune effector cells or T cells comprises an edited IL-lORa gene, wherein the edit is a DSB repaired by HEJ. In particular embodiments, an immune effector cell or T cell comprises an edited IL-lORa gene, wherein the edit is a DSB repaired by NHEJ. The edit may be in a coding sequence of the IL- 10Ra gene, preferably in exon 2 of the IL-lORa gene, and more preferably at SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of the IL-lORa gene. In particular embodiments, the edit is an insertion or deletion (INDEL) of 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 or more nucleotides in a coding sequence of the IL-lORa gene, preferably in exon 2 of the IL-lORa gene, more preferably at SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of the IL-lORa gene. In a preferred embodiment, the edit is a deletion of 1, 2, 3, or 4 nucleotides in the coding sequence of the IL-lORa gene, preferably in exon 2 of the IL- lORa gene, more preferably at SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of the IL-lORa gene.
In one embodiment, the edit is a deletion of about 1, 2, 3, or 4 nucleotides in a coding sequence of the IL-lORa gene, preferably in exon 2 of the IL-lORa gene, more preferably at SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of the IL-lORa gene. In a preferred
embodiment, the edit is a deletion of 1, 2, 3, or 4 nucleotides in the coding sequence of the IL- lORa gene, preferably in exon 2 of the IL-lORa gene, more preferably at SEQ ID NO: 13 (or SEQ ID NO: 15) in exon 2 of the IL-lORa gene.
In particular embodiments, a population of cells comprising immune effector cells or T cells comprises an edited IL-lORa gene comprising a donor repair template incorporated at a DSB repaired by HDR. The donor repair template may encode a FoxP3 polypeptide or polypeptide that increases or stabilizes FoxP3 expression, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
In particular embodiments, a population of cells comprising immune effector cells or T cells comprises an edited IL-lORa gene comprising a donor repair template comprising an IL- lORa gene or portion thereof and is designed to introduce one or more mutations in a genomic IL-lORa sequence such that a mutant IL-lORa gene product is expressed.
In particular embodiments, a population of cells comprising immune effector cells or T cells having one or more mutations in the IL-lORa gene that eliminate or substantial decrease IL-lORa expression is edited with a nuclease variant in the presence of a donor repair template designed to correct the one or more mutations and to increase or restore expression of IL-lORa.
In particular embodiments, T cells comprising one or more loss-of-function mutations, nonsense mutations, missense mutations, splice site mutations in the IL-lORa gene that eliminate or substantial decrease IL-lORa expression are edited with a nuclease variant in the presence of a donor repair template designed to correct the one or more mutations and to increase or restore expression of IL-lORa.
In one preferred embodiment, the donor template is designed such that a polynucleotide is inserted at a target site in the IL-lORa gene without substantially disrupting IL-lORa expression.
Illustrative examples of loss-of-function mutations in the IL-lORa gene that may be corrected by the genome edited compositions and methods contemplated here include, but are not limited to, W45G; Y64C; W69R; T84I; Y91C; V100G; R101W; Rl 17H; S138G; G141R; I169T; c.537G> A, p.T179T; g.IVS5+2T>C, c.690_765del, P206X; R262C, and E431X. In various embodiments, a genome edited cell comprises an edit in the IL-lORa gene and further comprises a polynucleotide encoding FoxP3, a bispecific T cell engager (BiTE) molecule; a cytokine (e.g., IL-2, insulin, IFN-γ, IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-a), a chemokine (e.g., ΜΙΡ-Ια, ΜΙΡ-Ιβ, MCP-1, MCP-3, and RANTES), a cytotoxin (e.g., Perforin, Granzyme A, and Granzyme B), a cytokine receptor (e.g., an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, and an IL-21 receptor), or an engineered antigen receptor (e.g., an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a Daric receptor or components thereof, or a chimeric cytokine receptor receptor). In one embodiment, a donor repair template comprising the polynucleotide and a nuclease variant are introduced into the cell and the polynucleotide is incorporated into the cell' s genome at the DSB site in the IL-lORa gene by HDR repair. The polynucleotide may also be introduced into the cell at a site other than the IL-lORa gene, e.g., by transducting the cell with a vector comprising the polynucleotide.
I. COMPOSITIONS AND FORMULATIONS
The compositions contemplated in particular embodiments may comprise one or more polypeptides, polynucleotides, vectors comprising same, and genome editing compositions and genome edited cell compositions, as contemplated herein. The genome editing compositions and methods contemplated in particular embodiments are useful for editing a target site in the human interleukin 10 receptor 1 alpha (IL-lORa) gene in a cell or a population of cells. In preferred embodiments, a genome editing composition is used to edit an IL-lORa gene in a hematopoietic cell, e.g., a T cell, an immune effector cell, or a Treg cell.
In various embodiments, the compositions contemplated herein comprise a nuclease variant, and optionally an end-processing enzyme, e.g., a 3 '-5' exonuclease (Trex2). The nuclease variant may be in the form of an mRNA that is introduced into a cell via
polynucleotide delivery methods disclosed supra, e.g., electroporation, lipid nanoparticles, etc. In one embodiment, a composition comprising an mRNA encoding a homing endonuclease variant or megaTAL, and optionally a 3 '-5' exonuclease, is introduced in a cell via
polynucleotide delivery methods disclosed supra. The composition may be used to generate a genome edited cell or population of genome edited cells by error prone NHEJ. In various embodiments, the compositions contemplated herein comprise a donor repair template. The composition may be delivered to a cell that expresses or will express nuclease variant, and optionally an end-processing enzyme. In one embodiment, the composition may be delivered to a cell that expresses or will express a homing endonuclease variant or megaTAL, and optionally a 3 '-5' exonuclease. Expression of the gene editing enzymes in the presence of the donor repair template can be used to generate a genome edited cell or population of genome edited cells by HDR.
In particular embodiments, the compositions contemplated herein comprise a population of cells, a nuclease variant, and optionally, a donor repair template. In particular embodiments, the compositions contemplated herein comprise a population of cells, a nuclease variant, an end-processing enzyme, and optionally, a donor repair template. The nuclease variant and/or end-processing enzyme may be in the form of an mRNA that is introduced into the cell via polynucleotide delivery methods disclosed supra.
In particular embodiments, the compositions contemplated herein comprise a population of cells, a homing endonuclease variant or megaTAL, and optionally, a donor repair template. In particular embodiments, the compositions contemplated herein comprise a population of cells, a homing endonuclease variant or megaTAL, a 3 '-5' exonuclease, and optionally, a donor repair template. The homing endonuclease variant, megaTAL, and/or 3 '-5 ' exonuclease may be in the form of an mRNA that is introduced into the cell via polynucleotide delivery methods disclosed supra.
In particular embodiments, the population of cells comprise genetically modified hematopoietic cells including, but not limited to, T cells, immune effector cells, and Tregs.
Compositions include, but are not limited to pharmaceutical compositions. A
"pharmaceutical composition" refers to a composition formulated in pharmaceutically- acceptable or physiologically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions may be administered in combination with other agents as well, such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the composition.
The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic cells are administered. Illustrative examples of pharmaceutical carriers can be sterile liquids, such as cell culture media, water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients in particular embodiments, include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
In one embodiment, a composition comprising a pharmaceutically acceptable carrier is suitable for administration to a subject. In particular embodiments, a
composition comprising a carrier is suitable for parenteral administration, e.g.,
intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration. In particular embodiments, a composition comprising a pharmaceutically acceptable carrier is suitable for intraventricular, intraspinal, or intrathecal administration.
Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media, or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is
incompatible with the transduced cells, use thereof in the pharmaceutical compositions is contemplated. In particular embodiments, compositions contemplated herein comprise genetically modified T cells and a pharmaceutically acceptable carrier. A composition comprising a cell-based composition contemplated herein can be administered separately by enteral or parenteral administration methods or in combination with other suitable compounds to effect the desired treatment goals.
The pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the human subject being treated. It further should maintain or increase the stability of the composition. The pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with other components of the composition. For example, the pharmaceutically acceptable carrier can be, without limitation, a binding agent {e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a filler {e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate, etc.), a lubricant {e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.), a disintegrant {e.g., starch, sodium starch glycolate, etc.), or a wetting agent {e.g., sodium lauryl sulfate, etc.). Other suitable pharmaceutically acceptable carriers for the compositions contemplated herein include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins, hydroxymethylcelluloses, polyvinylpyrrolidones and the like.
Such carrier solutions also can contain buffers, diluents and other suitable additives. The term "buffer" as used herein refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH. Examples of buffers contemplated herein include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), normal/physiologic saline (0.9% NaCl).
The pharmaceutically acceptable carriers may be present in amounts sufficient to maintain a pH of the composition of about 7. Alternatively, the composition has a pH in a range from about 6.8 to about 7.4, e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4. In still another embodiment, the composition has a pH of about 7.4.
Compositions contemplated herein may comprise a nontoxic pharmaceutically acceptable medium. The compositions may be a suspension. The term "suspension" as used herein refers to non-adherent conditions in which cells are not attached to a solid support. For example, cells maintained as a suspension may be stirred or agitated and are not adhered to a support, such as a culture dish.
In particular embodiments, compositions contemplated herein are formulated in a suspension, where the genome edited T cells are dispersed within an acceptable liquid medium or solution, e.g., saline or serum-free medium, in an intravenous (IV) bag or the like. Acceptable diluents include, but are not limited to water, PlasmaLyte, Ringer's solution, isotonic sodium chloride (saline) solution, serum-free cell culture medium, and medium suitable for cryogenic storage, e.g., Cryostor® medium.
In certain embodiments, a pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin, and suitable for storing a composition comprising a population of genome edited T cells. The therapeutic composition is intended to be administered into a human patient, and thus is substantially free of cell culture components such as bovine serum albumin, horse serum, and fetal bovine serum.
In some embodiments, compositions are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects. In particular embodiments, the pharmaceutically acceptable cell culture medium is a serum free medium.
Serum-free medium has several advantages over serum containing medium, including a simplified and better defined composition, a reduced degree of contaminants, elimination of a potential source of infectious agents, and lower cost. In various embodiments, the serum-free medium is animal-free, and may optionally be protein-free. Optionally, the medium may contain biopharmaceutically acceptable recombinant proteins. "Animal-free" medium refers to medium wherein the components are derived from non-animal sources. Recombinant proteins replace native animal proteins in animal- free medium and the nutrients are obtained from synthetic, plant or microbial sources. "Protein-free" medium, in contrast, is defined as substantially free of protein.
Illustrative examples of serum-free media used in particular compositions includes, but is not limited to QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO lO.
In a preferred embodiment, the compositions comprising genome edited T cells are formulated in PlasmaLyte.
In various embodiments, compositions comprising genome edited T cells are formulated in a cryopreservation medium. For example, cryopreservation media with cryopreservation agents may be used to maintain a high cell viability outcome post-thaw. Illustrative examples of cryopreservation media used in particular compositions includes, but is not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.
In one embodiment, the compositions are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS10.
In particular embodiments, the composition is substantially free of mycoplasma, endotoxin, and microbial contamination. By "substantially free" with respect to endotoxin is meant that there is less endotoxin per dose of cells than is allowed by the FDA for a biologic, which is a total endotoxin of 5 EU/kg body weight per day, which for an average 70 kg person is 350 EU per total dose of cells. In particular embodiments, compositions comprising hematopoietic stem or progenitor cells transduced with a retroviral vector contemplated herein contain about 0.5 EU/mL to about 5.0 EU/mL, or about 0.5 EU/mL, 1.0 EU/mL, 1.5 EU/mL, 2.0 EU/mL, 2.5 EU/mL, 3.0 EU/mL, 3.5 EU/mL, 4.0 EU/mL, 4.5 EU/mL, or 5.0 EU/mL.
In certain embodiments, compositions and formulations suitable for the delivery of polynucleotides are contemplated including, but not limited to, one or more mRNAs encoding one or more reprogrammed nucleases, and optionally end-processing enzymes.
Exemplary formulations for ex vivo delivery may also include the use of various transfection agents known in the art, such as calcium phosphate, electroporation, heat shock and various liposome formulations (i.e., lipid-mediated transfection). Liposomes, as described in greater detail below, are lipid bilayers entrapping a fraction of aqueous fluid. DNA spontaneously associates to the external surface of cationic liposomes (by virtue of its charge) and these liposomes will interact with the cell membrane.
In particular embodiments, formulation of pharmaceutically-acceptable carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., enteral and parenteral, e.g., intravascular, intravenous, intrarterial, intraosseously, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation. It would be understood by the skilled artisan that particular embodiments contemplated herein may comprise other formulations, such as those that are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy, volume I and volume II. 22nd Edition. Edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012, which is incorporated by reference herein, in its entirety.
J. GENOME EDITED CELL THERAPIES
The genome edited cells manufactured by the methods contemplated in particular embodiments provide improved drug products for use in the prevention, treatment, or amelioration of at least one symptom of a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency. As used herein, the term "drug product" refers to genetically modified cells produced using the compositions and methods contemplated herein. In particular embodiments, the drug product comprises genetically modified immune effector cells or T cells.
In particular embodiments, an effective amount of genome edited immune effector cells or T cells comprising an edited IL-lORa gene are administered to a subject to prevent, treat, or ameliorate at least one symptom of a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.
In particular embodiments, the IL-lORa edited cells do not substantially express, or lack expression of, IL-lORa and therefore lack or substantially lack functional IL-lORa expression, e.g., lack the ability to increase T cell exhaustion and to inhibit expression of MHC class II molecules, costimulatory molecules, and proinflammatory cytokines. In particular embodiments, genome edited immune effector cells that lack IL-lORa are more resistant to immunosuppressive signals from the tumor microenvironment.
In particular embodiments, a method of preventing, treating, or ameliorating at least one symptom of a cancer comprises administering the subject an effective amount of genome edited immune effector cells or T cells comprising an edited IL-lORa gene and an engineered TCR, CAR, or Daric, or other therapeutic transgene to redirect the cells to a tumor or cancer. The genetically modified cells are a more durable and persistant drug product because the cells are more resistant to immunosuppressive signals from the tumor microenvironment by virtue of editing the IL-lORa gene to decrease or eliminate IL-lORa expression.
In particular embodiments, genome edited cells contemplated herein are used in the treatment of solid tumors or cancers.
In particular embodiments, genome edited cells contemplated herein are used in the treatment of solid tumors or cancers including, but not limited to: adrenal cancer,
adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical
teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, bronchial tumors, cardiac tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer,
craniopharyngioma, ductal carcinoma in situ (DCIS) endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous histiosarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), germ cell tumors, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular cancer, hypopharyngeal cancer, intraocular melanoma, kaposi sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, menangioma, melanoma, Merkel cell carcinoma, midline tract carcinoma, mouth cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer,
nasopharyngeal cancer, neuroblastoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell tumors, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, stomach cancer, sweat gland carcinoma, synovioma, testicular cancer, throat cancer, thymus cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms Tumor.
In particular embodiments, genome edited cells contemplated herein are used in the treatment of solid tumors or cancers including, without limitation, liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.
In particular embodiments, genome edited cells contemplated herein are used in the treatment of various cancers including but not limited to pancreatic, bladder, and lung.
In particular embodiments, genome edited cells contemplated herein are used in the treatment of liquid cancers or hematological cancers.
In particular embodiments, genome edited cells contemplated herein are used in the treatment of B-cell malignancies, including but not limited to: leukemias, lymphomas, and multiple myeloma.
In particular embodiments, genome edited cells contemplated herein are used in the treatment of liquid cancers including, but not limited to leukemias, lymphomas, and multiple myelomas: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic, promyelocyte, myelomonocytic, monocytic, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sezary syndrome, precursor T- lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.
In particular embodiments, an effective amount of T cells comprising an IL-lORa gene edited using HDR to restore IL-lORa expression and/or increase or stabilize expression and/or function of FoxP3 or a polypeptide that enhances development, stability, and/or functionality of Treg cells is administered to a subject to prevent, treat, or ameliorate at least one symptom of GVHD, transplant rejection, an autoimmune disease or an inflammatory disease. In one embodiment, the genome edited cells are regulatory T cells (Tregs). Restoring IL-lORa expression in Treg cells would restore the cells' function of maintaining immune tolerance and immune system homeostasis. Enhancing FoxP3 function in Tregs is contemplated to enhance development, stability, and/or functionality of Treg cells.
Illustrative examples of diseases treated with genome edited Treg cells comprising an IL-lORa gene edited to restore IL-lORa expression and/or increase or stabilize expression and/or function of FoxP3 or a polypeptide that enhances development, stability, and/or functionality of Treg cells include, but are not limited to: Hashimoto' s thyroiditis, Grave' s disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, GVHD, transplant rejection, arthritis, and inflammatory bowel disease (IBD).
In particular embodiments, the GVHD is associated with solid organ transplants. In particular embodiments, an individual administered the genome edited Treg cells contemplated herein as received or is a candidate to receive a solid organ transplant. In certain embodiments, the solid organ transplant is selected from the group consisting of: a heart transplant, a lung transplant, a kidney transplant, a pancreas transplant, and a liver transplant.
In particular embodiments, the individual is administered the genome edited Treg cells contemplated herein to decrease GVHD while simultaneously maintaining or augmenting a GVL response post-transplant, e.g., bone marrow transplant. Allogeneic lymphocytes produce a strong graft-versus-leukemia (GVL) effect, but the beneficial effect is limited by graft-versus- host disease (GVHD). Particular embodiments, contemplate that administration of the genome edited Tregs will produce a GVL effect while suppressing GVHD. Illustrative examples of IBD treated with genome edited Treg cells comprising an IL- lORa gene edited to restore IL-lORa expression and/or increase or stabilize expression and/or function of FoxP3 or a polypeptide that enhances development, stability, and/or functionality of Treg cells include, but are not limited to: ulcerative colitis, early onset ulcerative colitis, very early onset ulcerative colitis, pancolitis, Crohn's disease, and neonatal-onset Crohn's disease.
In particular embodiments, an effective amount of T cells comprising an IL-lORa gene edited to maintain or restore IL-lORa expression and introduce a polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells is administered to a subject to prevent, treat, or ameliorate at least one symptom of GVHD, an autoimmune disease, an inflammatory disease, or an immunodeficiency. In one embodiment, the polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells is inserted into the IL-lORa gene at DSB introduced by a nuclease variant and repaired by HDR Without wishing to be bound by any particular theory, it is believe that increased FoxP3 expression in particular T cells can induce and/or stabilize a Treg phenotype.
Illustrative examples of diseases treated with genome edited Treg cells comprising an IL-lORa gene edited to maintain or restore IL-lORa expression and introduce a polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells include, but are not limited to:
GVHD, transplant rejection, arthritis, and inflammatory bowel disease (IBD).
Illustrative examples of IBD treated with genome edited Treg cells comprising an IL- lORa gene edited to maintain or restore IL-lORa expression and introduce a polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells include, but are not limited to:
ulcerative colitis, early onset ulcerative colitis, very early onset ulcerative colitis, pancolitis, Crohn's disease, and neonatal-onset Crohn's disease.
In various embodiments, the Tregs are edited with a polynucleotide encoding an exogenous promoter operably linked to a polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
In various embodiments, the Tregs are edited with a polynucleotide encoding a T2A or other viral self-cleaving peptide fused or linked to a polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
Preferred cells for use in the genome editing methods contemplated herein include autologous/autogeneic ("self) cells, preferably hematopoietic cells, more preferably T cells, and more preferably immune effector cells or Treg cells.
In particular embodiments, methods comprising administering a therapeutically effective amount of genome edited cells contemplated herein or a composition comprising the same, to a patient in need thereof, alone or in combination with one or more therapeutic agents, are provided. In certain embodiments, the cells are used in the treatment of patients at risk for developing a cancer, GVHD, transplant rejection, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency. Thus, particular embodiments comprise the treatment or prevention or amelioration of at least one symptom of a a cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency comprising administering to a subject in need thereof, a therapeutically effective amount of the genome edited cells contemplated herein.
In one embodiment, a method of treating a cancer, GVHD, transplant rejection, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency in a subject in need thereof comprises administering an effective amount, e.g., therapeutically effective amount of a composition comprising genome edited cells contemplated herein. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
In one illustrative embodiment, the effective amount of genome edited cells provided to a subject is at least 2 x 106 cells/kg, at least 3 x 106 cells/kg, at least 4 x 106 cells/kg, at least 5 x 106 cells/kg, at least 6 x 106 cells/kg, at least 7 x 106 cells/kg, at least 8 x 106 cells/kg, at least 9 x 106 cells/kg, or at least 10 x 106 cells/kg, or more cells/kg, including all intervening doses of cells.
In another illustrative embodiment, the effective amount of genome edited cells provided to a subject is about 2 x 106 cells/kg, about 3 x 106 cells/kg, about 4 x 106 cells/kg, about 5 x 106 cells/kg, about 6 x 106 cells/kg, about 7 x 106 cells/kg, about 8 x 106 cells/kg, about 9 x 106 cells/kg, or about 10 x 106 cells/kg, or more cells/kg, including all intervening doses of cells.
In another illustrative embodiment, the effective amount of genome edited cells provided to a subject is from about 2 x 106 cells/kg to about 10 x 106 cells/kg, about 3 x 106 cells/kg to about 10 x 106 cells/kg, about 4 x 106 cells/kg to about 10 x 106 cells/kg, about 5 x 106 cells/kg to about 10 x 106 cells/kg, 2 x 106 cells/kg to about 6 x 106 cells/kg, 2 x 106 cells/kg to about 7 x 106 cells/kg, 2 x 106 cells/kg to about 8 x 106 cells/kg, 3 x 106 cells/kg to about 6 x 106 cells/kg, 3 x 106 cells/kg to about 7 x 106 cells/kg, 3 x 106 cells/kg to about 8 x 106 cells/kg, 4 x 106 cells/kg to about 6 x 106 cells/kg, 4 x 106 cells/kg to about 7 x 106 cells/kg, 4 x 106 cells/kg to about 8 x 106 cells/kg, 5 x 106 cells/kg to about 6 x 106 cells/kg, 5 x 106 cells/kg to about 7 x 106 cells/kg, 5 x 106 cells/kg to about 8 x 106 cells/kg, or 6 x 106 cells/kg to about 8 x 106 cells/kg, including all intervening doses of cells.
One of ordinary skill in the art would recognize that multiple administrations of the compositions contemplated in particular embodiments may be required to effect the desired therapy. For example a composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more.
In certain embodiments, it may be desirable to administer activated T cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain embodiments, T cells can be activated from blood draws of from lOcc to 400cc. In certain embodiments, T cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, lOOcc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more. Not to be bound by theory, using this multiple blood draw/multiple reinfusion protocol may serve to select out certain populations of T cells. The administration of the compositions contemplated in particular embodiments may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, compositions are administered parenterally. The phrases "parenteral administration" and "administered parenterally" as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.
In one embodiment, a method of treating a subject diagnosed with a cancer, comprises removing immune effector cells from the subject, editing the genome of said immune effector cells and producing a population of genome edited immune effector cells, and administering the population of genome edited immune effector cells to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.
The methods for administering the cell compositions contemplated in particular embodiments include any method which is effective to result in reintroduction of ex vivo genome edited immune effector cells or on reintroduction of the genome edited progenitors of immune effector cells that on introduction into a subject differentiate into mature immune effector cells. One method comprises genome editing peripheral blood T cells ex vivo and returning the transduced cells into the subject.
All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference.
Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings contemplated herein that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.
EXAMPLES
EXAMPLE 1
REPROGRAMMING I-ONUI TO TARGET THE HUMAN IL-10Ra GENE
I-Onul was reprogrammed to target exon 2 of the IL-lORa gene by constructing modular libraries containing variable amino acid residues in the DNA recognition interface.
To construct the variants, degenerate codons were incorporated into I-Onul DNA binding domains using oligonucleotides. The oligonucleotides encoding the degenerate codons were used as PCR templates to generate variant libraries by gap recombination in the yeast strain S. cerevisiae. Each variant library spanned either the N- or C-terminal I-Onul DNA recognition domain and contained ~107 to 108 unique transformants. The resulting surface display libraries were screened by flow cytometry for cleavage activity against target sites comprising the corresponding domains "half-sites" (SEQ ID NOs: 16-17). Figure 2.
Yeast displaying the N- and C-terminal domain reprogrammed I-Onul HEs were purified and the plasmid DNA was extracted. PCR reactions were performed to amplify the reprogrammed domains, which were subsequently transformed into S. cerevisiae to create a library of reprogrammed domain combinations. Fully reprogrammed I-Onul variants that recognize the complete target site (SEQ ID NO: 13) present in exon 2 of the IL-lORa gene were identified from this library and purified.
EXAMPLE 2 REPROGRAMMED I-ONUI HOMING ENDONUCLEASES THAT EFFICIENTLY TARGET
EXON 2 OF THE IE- 1 ORa GENE
The activity of reprogrammed I-Onul HEs that target exon 2 of the IL-lORa gene was measured using a chromosomally integrated fluorescent reporter system (Certo et. al, 2011). Fully reprogrammed I-Onul HEs that bind and cleave the IL-lORa target sequence were cloned into mammalian expression plasmids and then individually transfected into a HEK 293T fibroblast cell line that was reprogrammed to contain the IL-lORa target sequence upstream of an out-of-frame gene encoding the fluorescent mCherry protein. Cleavage of the embedded target site by the HE and the subsequent accumulation of small insertions or deletions, caused by DNA repair via the non-homologous end joining (NHEJ) pathway, results in approximately one out of three repaired loci placing the fluorescent reporter gene back "in-frame". mCherry fluorescence is therefore a readout of endonuclease activity at the chromosomally embedded target sequence. The fully reprogrammed I-Onul HEs that bind and cleave the IL-lORa target site showed a moderate efficiency of mCherry expression in a cellular chromosomal context. Figure 3.
A secondary I-Onul variant library was generated by performing random mutagenesis on one of the reprogrammed I-Onul HEs that targets the IL-lORa target site, identified in the initial reporter screen (IL-10Ra.G7, SEQ ID NO: 6). In addition, display-based flow sorting was performed under more stringent affinity conditions (50 pM) to isolate variants with improved binding characteristics. Figure 3. This process identified an I-Onul variant, IL- lORa. G7. A3 (SEQ ID NO: 7), which has an approximately 2-fold higher rate of mCherry expressing cells than the parental I-Onul variant. Figure 3 (middle panel). Random
mutagenesis was performed on the I-Onul variant, IL-lORa. G7. A3 under more stringent cleavage conditions (pH of 6.8) to isolate variants with improved cleavage activity. This process identified an I-Onul variant, IL-10Ra.G7.A3.G7 (SEQ ID NO: 8), which has an approximately 33% higher rate of mCherry expressing cells than the parental I-Onul variant. Figure 3 (lower panel). IL-10Ra.G7.A3.G7 has subnanomolar affinity for the exon 2 target site (Figure 4). Figure 5 shows the relative alignments of representative I-Onul variants as well as the positional information of the residues comprising the DNA recognition interface.
EXAMPLE 3
EFFICIENT DISRUPTION OF EXON 2 OF THE IE- 1 ORa GENE
The I-Onul variant IL-10Ra.G7.A3.G7 was formatted as a megaTAL by appending an N-terminal 10.5 TAL array (SEQ ID NOs: 11 and 19) corresponding to an 11 base pair TAL array target site upstream of the IL-lORa LHE variant target site (SEQ ID NO: 14), using methods described in Boissel etal., 2013. Figure 6A. Another version of the megaTAL comprises a C-terminal fusion to Trex2 via a linker sequence (SEQ ID NO: 12). IL-10Ra.G7.A3.G7 megaTAL mRNA was prepared by in vitro transcription and co- transcriptionally capped with Anti-Reverse Cap Analog (ARC A) and enzymatically polyadenylated with poly(A) polymerase. The mRNA was purified and used to measure IL- 10Ra.G7.A3.G7 editing efficiency in primary human T cells.
Primary human Peripheral blood mononuclear cells (PBMC) were activated with anti-
CD3 and anti-CD28 antibodies and cultured in the presence of 250U/mL IL-2. At 3 days post- activation cells were electroporated with IL-10Ra.G7.A3.G7 megaTAL mRNA (SEQ ID NO: 19) in combination with Trex2 exonuclease (SEQ ID NO: 20).
Transfected T cells were expanded for additional 7-10 days and editing efficiency was measured using sequencing across the IL-lORa target site. The frequency of small insertion/deletion (indel) events across the IL-lORa target site was measured using Tracking of Indels by DEcomposition (TIDE, see Brinkman et al., 2014). Figure 6B shows a
representative TIDE analysis and illustrates the predominance of -1, -2, -3, or -4 indels at the target site of the IL-lORa megaTAL. EXAMPLE 4
IL10RA.G7.A3.G7 MEGATAL EFFICIENTLY DRIVES HOMOLOGY DIRECTED REPAIR
Adeno-associated virus (AAV) plasmids containing transgene cassettes comprising a promoter, a transgene encoding a fluorescent protein, and a polyadenylation signal (SEQ ID NO: 22) were designed and constructed. The integrity of AAV ITR elements was confirmed with Xmal digest. The transgene cassette was placed between two 300bp homology regions flanking the ILlORa megaTAL cleavage site (SEQ ID NO: 15). Neither homology region contained the complete megaTAL target site. Exemplary expression cassettes contain myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer- binding site substituted (MND) promoter operably linked to a polynucleotide encoding a fluorescent polypeptide, e.g., blue fluorescent protein (BFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), etc., and a WPRE
polyadenylation signal Figure 7A. megaTAL-induced homologous recombination was evaluated in primary human T cells activated with CD3 and CD28 and cultured in complete media supplemented with JL-2. After 3 days, T cells were washed and electroporated with in vitro transcribed mRNA encoding the IL10Roc.G7.A3.G7 megaTAL (SEQ ID NO: 19), and subsequently transduced with purified recombinant AAV encoding MND-GFP transgene cassette (SEQ ID NO: 22). Flow cytometry was used at multiple time points to measure the frequency of T cells expressing the fluorescent protein and to differentiate transient expression of the fluorescent protein from the non-integrated rAAV targeting vector.
Long-term transgene expression was observed in 35-65% of the T cells that were treated with both the megaTAL and the rAAV targeting vector. In untreated control samples, there was no fluorescent protein expression consistent with a lack of integration into the genome (Figure 7B). Results were confirmed in experiments performed on T cells isolated from independent donors. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

CLAIMS What is claimed is:
1. A polypeptide comprising a homing endonuclease (HE) variant that cleaves a target site in the human interleukin 10 receptor 1 alpha (IL-lORa) gene.
2. The polypeptide of claim 1, wherein the HE variant is an LAGLIDADG homing endonuclease (LHE) variant.
3. The polypeptide of claim 1, or claim 2, wherein the polypeptide comprises a biologically active fragment of the HE variant.
4. The polypeptide of claim 3, wherein the biologically active fragment lacks the 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal amino acids compared to a corresponding wild type HE.
5. The polypeptide of claim 4, wherein the biologically active fragment lacks the 4 N-terminal amino acids compared to a corresponding wild type HE.
6. The polypeptide of claim 4, wherein the biologically active fragment lacks the 8 N-terminal amino acids compared to a corresponding wild type HE.
7. The polypeptide of claim 3, wherein the biologically active fragment lacks the 1, 2, 3, 4, or 5 C-terminal amino acids compared to a corresponding wild type HE.
8. The polypeptide of claim 7, wherein the biologically active fragment lacks the C- terminal amino acid compared to a corresponding wild type HE.
9. The polypeptide of claim 7, wherein the biologically active fragment lacks the 2 C-terminal amino acids compared to a corresponding wild type HE.
10. The polypeptide of any one of claims 1 to 9, wherein the HE variant is a variant of an LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, I-ApaMI, I-CapIII, I- CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I- EjeMI, I-GpeMI, I-Gpil, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-Ltrl, I-LtrWI, I- MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-Onul, I-OsoMI, I-OsoMII, I-OsoMIII, I- OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, and I-Vdil41I.
11. The polypeptide of any one of claims 1 to 10, wherein the HE variant is a variant of an LHE selected from the group consisting of: I-CpaMI, I-HjeMI, I-Onul, I-PanMI, and SmaMI.
12. The polypeptide of any one of claims 1 to 11, wherein the HE variant is an I-Onul LHE variant.
13. The polypeptide of any one of claims 1 to 12, wherein the HE variant comprises one or more amino acid substitutions in the DNA recognition interface at amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
14. The polypeptide of any one of claims 1 to 13, wherein the HE variant comprises at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions in the DNA recognition interface at amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 of an l-OnuI LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
15. The polypeptide of any one of claims 1 to 13, wherein the HE variant comprises at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions in at least one position selected from the position group consisting of positions: 24, 26, 28, 30, 32, 34, 36, 37, 38, 40, 41, 42, 44, 46, 48, 59, 70, 72, 75, 78, 80, 82, 138, 143, 145, 159, 168, 180, 182, 184, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 207, 223, 225, 227, 228, 229, 232, 236, 238, and 240 of any one of SEQ ID NOs: 1-5, or a biologically active fragment thereof.
16. The polypeptide of any one of claims 1 to 14, wherein the HE variant comprises at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46V, Q46I, T48G, N59S, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180H, C180Y, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K, and V238I, in reference to an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
17. The polypeptide of any one of claims 1 to 15, wherein the HE variant comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46V, T48G, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, S159P, F168L, C180H, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, K229A, F232K, D236K, and V238I, in reference to an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
18. The polypeptide of any one of claims 1 to 15, wherein the HE variant comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46I, T48G, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180H, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K, and V238I, in reference to an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
19. The polypeptide of any one of claims 1 to 15, wherein the HE variant comprises the following amino acid substitutions: S24C, L26S, R28S, R30Y, N32T, K34R, S36R, V37A, G38R, S40R, E42R, G44S, Q46I, T48G, N59S, A70T, S72A, N75G, S78Q, K80R, T82S, L138M, T143N, E145K, S159P, F168L, C180Y, F182Y, N184R, S188P, K189R, S190R, K191D, L192A, G193R, Q195Y, Q197G, V199G, S201E, T203G, K207R, Y223R, K225Y, K227Q, N228K, K229A, F232K, D236K, and V238I, in reference to an I-Onul LHE amino acid sequence as set forth in SEQ ID NOs: 1-5, or a biologically active fragment thereof.
20. The polypeptide of any one of claims 1 to 19, wherein the HE variant comprises an amino acid sequence that is at least 80%, preferably at least 85%>, more preferably at least 90%), or even more preferably at least 95%> identical to the amino acid sequence set forth in any one of SEQ ID NOs: 6-8, or a biologically active fragment thereof.
21. The polypeptide of any one of claims 1 to 20, wherein the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.
22. The polypeptide of any one of claims 1 to 20, wherein the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 7, or a biologically active fragment thereof.
23. The polypeptide of any one of claims 1 to 20, wherein the HE variant comprises the amino acid sequence set forth in SEQ ID NO: 8, or a biologically active fragment thereof.
I l l
24. The polypeptide of any one of claims 1 to 23, further comprising a DNA binding domain.
25. The polypeptide of claim 24, wherein the DNA binding domain is selected from the group consisting of: a TALE DNA binding domain and a zinc finger DNA binding domain.
26. The polypeptide of claim 25, wherein the TALE DNA binding domain comprises about 9.5 TALE repeat units to about 15.5 TALE repeat units.
27. The polypeptide of claim 25 or claim 26, wherein the TALE DNA binding domain binds a polynucleotide sequence in the IL-lORa gene.
28. The polypeptide of any one of claims 25 to 27, wherein the TALE DNA binding domain binds the polynucleotide sequence set forth in SEQ ID NO: 11.
29. The polypeptide of claim 25, wherein the zinc finger DNA binding domain comprises 2, 3, 4, 5, 6, 7, or 8 zinc finger motifs.
30. The polypeptide of any one of claims 1 to 29 further comprising a peptide linker and an end-processing enzyme or biologically active fragment thereof.
31. The polypeptide of any one of claims 1 to 29, further comprising a viral self- cleaving 2A peptide and an end-processing enzyme or biologically active fragment thereof.
32. The polypeptide of claim 30 or claim 31, wherein the end-processing enzyme or biologically active fragment thereof has 5 '-3 ' exonuclease, 5 '-3' alkaline exonuclease, 3 '-5' exonuclease, 5' flap endonuclease, helicase or template-independent DNA polymerase activity.
33. The polypeptide of any one of claims 30 to 32, wherein the end-processing enzyme comprises Trex2 or a biologically active fragment thereof.
34. The polypeptide of any one of claims 1 to 33, wherein the polypeptide cleaves the human IL-lORa gene at the polynucleotide sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12.
35. A polynucleotide encoding the polypeptide of any one of claims 1 to 34.
36. An mRNA encoding the polypeptide of any one of claims 1 to 34.
37. A cDNA encoding the polypeptide of any one of claims 1 to 34.
38. A vector comprising a polynucleotide encoding the polypeptide of any one of claims 1 to 34.
39. A cell comprising the polypeptide of any one of claims 1 to 34.
40. A cell comprising a polynucleotide encoding the polypeptide of any one of claims
1 to 34.
41. A cell comprising the vector of claim 38.
42. A cell comprising one or more genome modifications introduced by the polypeptide of any one of claims 1 to 34.
43. The cell of any one of claims 39 to 42, wherein the cell is a hematopoietic cell.
44. The cell of any one of claims 39 to 43, wherein the cell is a T cell.
45. The cell of any one of claims 39 to 44, wherein the cell is a CD3+, CD4+, and/or CD8+ cell.
46. The cell of any one of claims 39 to 45, wherein the cell is an immune effector cell.
47. The cell of any one of claims 39 to 46, wherein the cell is a cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cells.
48. The cell of any one of claims 39 to 46, wherein the cell is a natural killer (NK) cell or natural killer T ( KT) cell.
49. The cell of any one of claims 39 to 45, wherein the cell is a regulatory T cell
(Treg).
50. The cell of any one of claims 39 to 49, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
51. The cell of any one of claims 39 to 50, wherein the cell comprises a
polynucleotide encoding an engineered antigen receptor.
52. The cell of claim 51, wherein the engineered antigen receptor is selected from the group consisting of: an engineered T cell receptor, a chimeric antigen receptor, a DARIC, or a zetakine.
53. The cell of any one of claims 39 to 50, wherein the cell comprises a
polynucleotide encoding a bispecific T cell engager (BiTE) molecule; a hormone; a cytokine (e.g., IL-2, insulin, IFN-γ, IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-a), a chemokine (e.g., ΜΙΡ-Ια, ΜΙΡ-Ιβ, MCP-1, MCP-3, and RANTES), a cytotoxin (e.g., Perforin, Granzyme A, and Granzyme B), or a cytokine receptor (e.g., an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, or an IL-15 receptor, and an IL-21 receptor).
54. The cell of any one of claims 39 to 50, wherein the cell comprises a
polynucleotide encoding FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
55. The cell of any one of claims 51 to 54, where the polynucleotide is integrated into the IL-l ORa gene.
56. The cell of any one of claims 49 to 55, where the polynucleotide is a donor repair template integrated into the IL-lORa gene at a DNA double stranded break site introduced by the polypeptide according to any one of claims 1 to 34.
57. The cell of any one of claims 49 to 56, wherein the polynucleotide is a donor repair template designed to correct one or more loss-of-function mutations in the endogenous IL- lORa gene, and wherein the donor repair template is integrated into the IL-lORa gene at a DNA double stranded break site introduced by the polypeptide according to any one of claims 1 to 34.
58. The cell of claim 56 or claim 57, wherein IL-lORa expression is maintained, restored or increased and the polynucleotide encodes FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
59. A plurality of cells comprising one or more cells of any one of claims 39 to 58.
A composition comprising one or more cells according to any one of claims 39 to
61. A composition comprising one or more cells according to any one of claims 39 to 59 and a physiologically acceptable carrier.
62. A method of editing a human IL-lORa gene in a cell comprising: introducing a polynucleotide encoding the polypeptide of any one of claims 1 to 34 into the cell, wherein expression of the polypeptide creates a double strand break at a target site in a human IL-lORa gene.
63. A method of editing a human IL-lORa gene in cell comprising: introducing a polynucleotide encoding the polypeptide of any one of claims 1 to 34 into the cell, wherein expression of the polypeptide creates a double strand break at a target site in a human IL-lORa gene, wherein the break is repaired by non-homologous end joining ( HEJ).
64. A method of editing a human IL-lORa gene in a cell comprising: introducing a polynucleotide encoding the polypeptide of any one of claims 1 to 34 and a donor repair template into the cell, wherein expression of the polypeptide creates a double strand break at a target site in a human IL-lORa gene and the donor repair template is incorporated into the human IL-lORa gene by homology directed repair (HDR) at the site of the double-strand break (DSB).
65. The method of claim 64, wherein IL-lORa expression is maintained, restored or increased and the polynucleotide encodes FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances development, stability, and/or functionality of Treg cells.
66. The method of any one of claims 62 to 65, wherein the cell is a hematopoietic cell.
67. The method of any one of claims 62 to 66, wherein the cell is a T cell.
68. The method of any one of claims 62 to 67, wherein the cell is a CD3+, CD4+, and/or CD8+ cell.
69. The method of any one of claims 62 to 68, wherein the cell is an immune effector cell.
70. The method of any one of claims 62 to 69, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.
71. The method of any one of claims 62 to 69, wherein the cell is a natural killer (NK) cell or natural killer T (NKT) cell.
72. The method of any one of claims 62 to 68, wherein the cell is a regulatory T cell
(Treg).
73. The method of any one of claims 62 to 72, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
74. The method of any one of claims 62 to 73, wherein the polynucleotide encoding the polypeptide is an mRNA.
75. The method of any one of claims 62 to 74, wherein a polynucleotide encoding a 5 '-3 ' exonuclease is introduced into the cell.
76. The method of any one of claims 62 to 74, wherein a polynucleotide encoding Trex2 or a biologically active fragment thereof is introduced into the cell.
77. The method of any one of claims 64 to 76, wherein the donor repair template encodes a FoxP3, a polypeptide that increases FoxP3, or a polypeptide that enhances
development, stability, and/or functionality of Treg cells.
78. The method of any one of claims 64 to 76, wherein the donor repair template encodes a wild type copy of the IL-lORa gene or portion thereof.
79. The method of any one of claims 64 to 76, wherein the donor repair template encodes an IL-lORa gene or portion thereof comprising one or more mutations compared to the wild type IL-lORa gene.
80. The method of any one of claims 64 to 76, wherein the donor repair template encodes an engineered antigen receptor.
81. The method of claim 80, wherein the engineered antigen receptor is selected from the group consisting of: an engineered T cell receptor, a chimeric antigen receptor, a DARIC, or a zetakine.
82. The method of any one of claims 64 to 76, wherein the donor repair template encodes a bispecific T cell engager (BiTE) molecule; a hormone; a cytokine (e.g. , JL-2, insulin, IFN-γ, IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-a), a chemokine (e.g., ΜΙΡ-Ια, ΜΙΡ-Ιβ, MCP- 1, MCP-3, and RANTES), a cytotoxin (e.g., Perforin, Granzyme A, and Granzyme B), or a cytokine receptor (e.g. , an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, or an IL-15 receptor, and an IL-21 receptor).
83. The method of any one of claims 64 to 82, wherein the donor repair template comprises a 5 ' homology arm homologous to a human IL-lORa gene sequence 5 ' of the DSB and a 3 ' homology arm homologous to a human IL-lORa gene sequence 3 ' of the DSB.
84. The method of claim 83, wherein the lengths of the 5' and 3 ' homology arms are independently selected from about 100 bp to about 2500 bp.
85. The method of claim 83 or claim 84, wherein the lengths of the 5' and 3 ' homology arms are independently selected from about 600 bp to about 1500 bp.
86. The method of any one of claims 83 to 85, wherein the 5 'homology arm is about 1500 bp and the 3 ' homology arm is about 1000 bp.
87. The method of any one of claims 83 to 86, wherein the 5 'homology arm is about 600 bp and the 3 ' homology arm is about 600 bp.
88. The method of any one of claims 83 to 87, wherein a viral vector is used to introduce the donor repair template into the cell.
89. The method of claim 88, wherein the viral vector is a recombinant adeno- associated viral vector (rAAV) or a retrovirus.
90. The method of claim 89, wherein the rAAV has one or more ITRs from AAV2.
91. The method of claim 89 or claim 90, wherein the rAAV has a serotype selected from the group consisting of: AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVl 0.
92. The method of any one of claims 89 to 91, wherein the rAAV has an AAV2 or AAV6 serotype.
93. The method of claim 89, wherein the retrovirus is a lentivirus.
94. The method of claim 93, wherein the lentivirus is an integrase deficient lentivirus (TDLV).
95. A method of treating, preventing, or ameliorating at least one symptom of a cancer, GVHD, transplant rejection, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or condition associated therewith, comprising administering to the subject an effective amount of the composition of claim 60 or claim 61.
96. A method of treating a solid cancer comprising administering to the subject an effective amount of the composition of claim 60 or claim 61.
97. The method of claim 96, wherein the solid cancer comprises liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.
98. A method of treating a hematological malignancy comprising administering to the subject an effective amount of the composition of claim 60 or claim 61.
99. The method of claim 98, wherein the hematological malignancy is a leukemia, lymphoma, or multiple myeloma.
100. A method of treating an autoimmune disease comprising administering to the subject an effective amount of the composition of claim 60 or claim 61.
101. The method of claim 100, wherein the autoimmune disease is associated with a loss-of-function mutation in the IL-lORa gene.
102. The method of claim 101, wherein the loss-of-function mutation is a missense mutation, nonsense mutation, or splice site mutation.
103. The method of any one of claims 100 to 102, wherein the autoimmune disease is arthritis.
104. The method of any one of claims 100 to 102, wherein the autoimmune disease is inflammatory bowel disease (IBD).
105. The method of claim 104, wherein the IBD is selected from the group consisting of ulcerative colitis, early onset ulcerative colitis, very early onset ulcerative colitis, pancolitis, Crohn's disease, and neonatal-onset Crohn's disease.
106. The method of any one of claims 100 to 105, wherein the autoimmune disease is associated with a loss-of-function mutation in the IL-lORa gene selected from the group consisting of: W45G; Y64C; W69R; T84I; Y91C; V100G; R101W; R117H; S138G; G141R; I169T; c.537G > A, p.T179T; g.IVS5+2T>C, c.690_765del, P206X; R262C, and E431X.
107. A method of treating graft-versus-host disease (GVHD) comprising administering to the subject an effective amount of the composition of claim 60 or claim 61.
108. The method of claim 107, wherein the GVHD is associated with a solid organ transplant in the subject.
109. The method of claim 107 or claim 108, wherein the solid organ transplant is selected from the group consisting of: a heart transplant, a lung transplant, a kidney transplant, a pancreas transplant, and a liver transplant.
110. A method of preventing graft-versus-host disease (GVHD) while maintaining a graft-versus-leukemia response comprising administering to the subject an effective amount of the composition of claim 60 or claim 61.
111. The method of claim 110, wherein the GVHD is associated with a bone marrow transplant in the subject.
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