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• • C—CHEMISTRY; METALLURGY
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  • 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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
• • 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
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1241—Nucleotidyltransferases (2.7.7)
• • 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
  • C12N2510/00—Genetically modified cells
• • 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
  • C12N2800/00—Nucleic acids vectors
  • C12N2800/90—Vectors containing a transposable element
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
  • C12Y207/07—Nucleotidyltransferases (2.7.7)

Definitions

• the present invention is directed to compositions and methods for targeted gene modification.
• DNA transposons When compared with viral transduction of immune cells, such as T lymphocytes, delivery of transgenes via DNA transposons, such as piggyBac and Sleeping Beauty, offers significant advantages in ease of use, ability to delivery much larger cargo, speed to clinic and cost of production.
• the piggyBac DNA transposon offers additional advantages in giving long-term, high-level and stable expression of transgenes, and in being significantly less mutagenic than a retrovirus, being non-oncogenic and being fully reversible.
• Previous attempts to use DNA transposons to deliver transgenes to T cells have been unsuccessful at generating commercially viable products or manufacturing methods because the previous methods have been inefficient.
• the data provided herein demonstrate not only that decreasing the amount of DNA transposon introduced into the cell increased viability but also that this method increased the percentage of cells that harbored a transposition event, resulting in a viable commercial process and a viable commercial product. Thus, the methods of the disclosure demonstrate success where others have failed.
• the disclosure provides a nonviral method for the ex-vivo genetic modification of an immune cell comprising delivering to the immune cell, (a) a nucleic acid or amino acid sequence comprising a sequence encoding a transposase enzyme and (b) a recombinant and non-naturally occurring DNA sequence comprising a DNA sequence encoding a transposon.
• the method further comprises the step of stimulating the immune cell with one or more cytokine(s).
• the sequence encoding a transposase enzyme is an mRNA sequence.
• the mRNA sequence encoding a transposase enzyme may be produced in vitro.
• the sequence encoding a transposase enzyme is a DNA sequence.
• the DNA sequence encoding a transposase enzyme may be produced in vitro.
• the DNA sequence may be a cDNA sequence.
• the sequence encoding a transposase enzyme is an amino acid sequence.
• the amino acid sequence encoding a transposase enzyme may be produced in vitro.
• a protein sPBo may be delivered following pre-incubation with transposon DNA.
• the delivering step comprises electroporation or nucleofection of the immune cell.
• the step of stimulating the immune cell with one or more cytokine(s) occurs following the delivering step. Alternatively, or in addition, in certain embodiments, the step of stimulating the immune cell with one or more cytokine(s) occurs prior to the delivering step. In certain embodiments, the one or more cytokine(s) comprise(s) IL-2, IL-21, IL-7 and/or IL-15.
• the immune cell is an autologous immune cell.
• the immune cell may be a human immune cell and/or an autologous immune cell.
• the immune cell may be derived from a non-autologous source, including, but not limited to a primary cell, a cultured cell or cell line, an embryonic or adult stem cell, an induced pluripotent stem cell or a transdifferentiated cell.
• the immune cell may have been previously genetically modified or derived from a cell or cell line that has been genetically modified.
• the immune cell may be modified or may be derived from a cell or cell line that has been modified to suppress one or more apoptotic pathways.
• the immune cell may be modified or may be derived from a cell or cell line that has been modified to be "universally" allogenic by a majority of recipients in the context, for example, of a therapy involving an adoptive cell transfer.
• the immune cell is an activated immune cell.
• the immune cell is an resting immune cell.
• the immune cell is a T- lymphocyte.
• the T-lymphocyte is an activated T-lymphocyte.
• the T-lymphocyte is a resting T-lymphocyte.
• the immune cell is a Natural Killer (NK) cell.
• NK Natural Killer
• the immune cell is a Cytokine-induced Killer (CIK) cell.
• CIK Cytokine-induced Killer
• the immune cell is a Natural Killer T (NKT) cell.
• the immune cell is isolated or derived from a human.
• the immune cell is isolated or derived from a non-human mammal.
• the non-human mammal is a rodent, a rabbit, a cat, a dog, a pig, a horse, a cow, or a camel.
• the immune cell is isolated or derived from a non-human primate.
• the transposase enzyme is a
• the Super piggyBac (sPBo) transposase enzyme may comprise or consist of an amino acid sequence at least 75% identical to:
• the transposase enzyme is a Sleeping Beauty transposase enzyme (see, for example, US Patent No. 9,228,180, the contents of which are incorporated herein in their entirety).
• the Sleeping Beauty transposase is a hyperactive Sleeping Beauty SB100X transposase.
• the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75% identical to:
• the Sleeping Beauty transposase is a hyperactive Sleeping Beauty SBlOOX transposase
• the Sleeping Beauty transposase is a hyperactive Sleeping Beauty SBlOOX transposase
• Beauty transposase enzyme comprises an amino acid sequence at least 75% identical to:
• ATKY (SEQ ID NO: 3).
• the recombinant and non- naturally occurring DNA sequence comprising a DNA sequence encoding a transposon may be circular.
• the DNA sequence encoding a transposon may be a plasmid vector.
• the DNA sequence encoding a transposon may be a minicircle DNA vector.
• the recombinant and non- naturally occurring DNA sequence encoding a transposon may be linear.
• the linear recombinant and non-naturally occurring DNA sequence encoding a transposon may be produced in vitro.
• Linear recombinant and non-naturally occurring DNA sequences of the disclosure may be a product of a restriction digest of a circular DNA.
• the circular DNA is a plasmid vector or a minicircle DNA vector.
• Linear recombinant and non-naturally occurring DNA sequences of the disclosure may be a product of a polymerase chain reaction (PCR).
• Linear recombinant and non-naturally occurring DNA sequences of the disclosure may be a double-stranded doggyboneTM DNA sequence.
• DoggyboneTM DNA sequences of the disclosure may be produced by an enzymatic process that solely encodes an antigen expression cassette, comprising antigen, promoter, poly-A tail and telomeric ends.
• the recombinant and non- naturally occurring DNA sequence encoding a transposon further comprises a sequence encoding a chimeric antigen receptor or a portion thereof.
• Chimeric antigen receptors (CARs) of the disclosure may comprise (a) an ectodomain comprising an antigen recognition region, (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain.
• the ectodomain may further comprise a signal peptide.
• the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
• the signal peptide may comprise a sequence encoding a human CD2, CD35, CD3s, CD3y, CO3C,, CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR signal peptide.
• the signal peptide may comprise a sequence encoding a human CD8a signal peptide.
• the transmembrane domain may comprise a sequence encoding a human CD2, CD35, CD3s, CD3y, CO3C,, CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain.
• the transmembrane domain may comprise a sequence encoding a human CD8a transmembrane domain.
• the endodomain may comprise a human CD3 ⁇ endodomain.
• the at least one costimulatory domain may comprise a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof.
• the at least one costimulatory domain may comprise a CD28 and/or a 4- IBB costimulatory domain.
• the hinge may comprise a sequence derived from a human CD8a, IgG4, and/or CD4 sequence. In certain embodiments of the CARs of the disclosure, the hinge may comprise a sequence derived from a human CD8a sequence.
• the recombinant and non- naturally occurring DNA sequence encoding a transposon further comprises a sequence encoding a chimeric antigen receptor or a portion thereof.
• the portion of the sequence encoding a chimeric antigen receptor may encode an antigen recognition region.
• the antigen recognition region may comprise one or more complementarity determining region(s).
• the antigen recognition region may comprise an antibody, an antibody mimetic, a protein scaffold or a fragment thereof.
• the antibody is a chimeric antibody, a recombinant antibody, a humanized antibody or a human antibody.
• the antibody is affinity -tuned.
• Nonlimiting examples of antibodies of the disclosure include a single-chain variable fragment (scFv), a VHH, a single domain antibody (sdAB), a small modular immunopharmaceutical (SMIP) molecule, or a nanobody.
• the VHH is camelid.
• the VHH is humanized.
• Nonlimiting examples of antibody fragments of the disclosure include a complementary determining region, a variable region, a heavy chain, a light chain, or any combination thereof.
• Nonlimiting examples of antibody mimetics of the disclosure include an affibody, an afflilin, an affimer, an affitin, an alphabody, an anticalin, and avimer, a DARPin, a Fynomer, a Kunitz domain peptide, or a monobody.
• Nonlimiting examples of protein scaffolds of the disclosure include a Centyrin.
• the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and an amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon is equal to or less than 10.0 ⁇ g per 100 of an electroporation or nucleofection reaction. In certain embodiments, a concentration of the amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 100 ⁇ g/mL.
• the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and an amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon is equal to or less than 7.5 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction. In certain embodiments, a concentration of the amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 75 ⁇ g/mL.
• the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and an amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon is equal to or less than 6.0 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction. In certain embodiments, a concentration of the amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 60 ⁇ g/mL.
• the transposase is a Sleeping Beauty transposase. In certain embodiments, the Sleeping Beauty transposase is a Sleeping Beauty 100X (SB100X) transposase.
• the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and an amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon is equal to or less than 5.0 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction. In certain embodiments, a concentration of the amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 50 ⁇ g/mL.
• the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and an amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon is equal to or less than 2.5 ⁇ g per 100 of an electroporation or nucleofection reaction. In certain embodiments, a concentration of the amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 25 ⁇ g/mL.
• the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and an amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon is equal to or less than 1.67 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction. In certain embodiments, a concentration of the amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 16.7 ⁇ g/mL. In certain embodiments, the transposase is a Super piggyBac (PB) transposase.
• PB Super piggyBac
• the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and an amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon is equal to or less than 0.55 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction. In certain embodiments, a concentration of the amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 5.5 ⁇ g/mL.
• the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and an amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon is equal to or less than 0.19 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction. In certain embodiments, a concentration of the amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 1.9 ⁇ g/mL.
• the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and an amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon is equal to or less than 0.10 ⁇ g per 100 of an electroporation or nucleofection reaction. In certain embodiments, a concentration of the amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 1.0 ⁇ g/mL.
• the nucleic acid sequence encoding the transposase enzyme is a RNA sequence
• an amount of the DNA sequence encoding the transposon is equal to or less than 10.0 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction.
• a concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 100 ⁇ g/mL.
• the nucleic acid sequence encoding the transposase enzyme is a RNA sequence
• an amount of the DNA sequence encoding the transposon is equal to or less than 7.5 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction.
• a concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 75 ⁇ g/mL.
• the nucleic acid sequence encoding the transposase enzyme is a RNA sequence
• an amount of the DNA sequence encoding the transposon is equal to or less than 6.0 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction.
• a concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 60 ⁇ g/mL.
• the transposase is a Sleeping Beauty transposase.
• the Sleeping Beauty transposase is a Sleeping Beauty 100X (SB100X) transposase.
• the nucleic acid sequence encoding the transposase enzyme is a RNA sequence
• an amount of the DNA sequence encoding the transposon is equal to or less than 5.0 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction.
• a concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 50 ⁇ g/mL.
• the nucleic acid sequence encoding the transposase enzyme is a RNA sequence
• an amount of the DNA sequence encoding the transposon is equal to or less than 2.5 ⁇ g per 100 of an electroporation or nucleofection reaction.
• a concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 25 ⁇ g/mL.
• the nucleic acid sequence encoding the transposase enzyme is a RNA sequence
• an amount of the DNA sequence encoding the transposon is equal to or less than 1.67 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction.
• a concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 16.7 ⁇ g/mL.
• the transposase is a Super piggyBac (PB) transposase.
• the nucleic acid sequence encoding the transposase enzyme is a RNA sequence
• an amount of the DNA sequence encoding the transposon is equal to or less than 0.55 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction.
• a concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 5.5 ⁇ g/mL.
• the nucleic acid sequence encoding the transposase enzyme is a RNA sequence
• an amount of the DNA sequence encoding the transposon is equal to or less than 0.19 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction.
• a concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 1.9 ⁇ g/mL.
• the nucleic acid sequence encoding the transposase enzyme is a RNA sequence
• an amount of the DNA sequence encoding the transposon is equal to or less than 1.0 ⁇ g per 100 ⁇ of an electroporation or nucleofection reaction.
• a concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 1.0 ⁇ g/mL.
• the disclosure provides an immune cell modified according to the method of the disclosure.
• the immune cell may be a T-lymphocyte, a Natural Killer (NK) cell, a Cytokine- induced Killer (CIK) cell or a Natural Killer T (NKT) cell.
• the immune cell may be further modified by a second gene editing tool, including, but not limited to those gene editing tools comprising an endonuclease operably-linked to either a Cas9 or a TALE sequence.
• the endonuclease is operably-linked to either a Cas9 or a TALE sequence covalently.
• the endonuclease is operably-linked to either a Cas9 or a TALE sequence non- covalently.
• the Cas9 is an inactivated Cas9 (dCas9).
• the inactivated Cas9 comprises D10A and N580A within the catalytic site.
• the Cas9 is a small and inactivated Cas9 (dSaCas9).
• the dSaCas9 comprises the amino acid sequence of
• the disclosure provides an immune cell modified according to the method of the disclosure.
• the immune cell may be a T-lymphocyte, a Natural Killer (NK) cell, a Cytokine- induced Killer (CIK) cell or a Natural Killer T (NKT) cell.
• the immune cell may be further modified by a second gene editing tool, including, but not limited to those gene editing tools comprising an endonuclease operably-linked to either a Cas9 or a TALE sequence.
• the second gene editing tool may include an excision-only piggyBac transposase to re-excise the inserted sequences or any portion thereof.
• the excision-only piggyBac transposase may be used to "re-excise" the transposon.
• the disclosure provides a composition comprising the immune cell of the disclosure.
• the disclosure provides a use of a composition comprising the immune cell of the disclosure for the treatment of a disease or disorder in a subject in need thereof.
• the disease or disorder is a cancer.
• the disease or disorder is an infectious disease.
• the infectious disease may be caused by a virus, bacterium, yeast, microbe or any combination thereof.
• the immune cell of the composition is autologous.
• the immune cell of the composition is allogeneic.
• the disclosure provides a culture media for enhancing viability of a modified immune cell comprising IL-2, IL-21, IL-7, IL-15 or any combination thereof.
• the modified immune cell may be a T-lymphocyte, a Natural Killer (NK) cell, a Cytokine-induced Killer (CIK) cell or a Natural Killer T (NKT) cell.
• the modified immune cell may contain one or more exogenous DNA sequences.
• the modified immune cell may contain one or more exogenous RNA sequences.
• the modified immune cell may have been electroporated or nucleofected.
• Figure 1 is a series of graphs depicting transfection efficiency and cell viability following plasmid DNA nucleofection in primary human T lymphocytes.
• Figure 2 is a series of graphs depicting DNA cytotoxicity to T cells.
• Figure 3 is a series of graphs showing that DNA-mediated cytotoxicity in T cells is dose dependent.
• Figure 4 is a series of graphs showing that extracellular plasmid DNA is not cytotoxic.
• Figure 5 is a series of graphs depicting efficient transposition using sPBo mRNA in Jurkat cells.
• Figure 6 is a series of graphs depicting efficient transposition in T lymphocytes using sPBo mRNA
• Figure 7 is a series of graphs depicting efficient delivery of linearized DNA transposon products.
• Figure 8 is a series of graphs showing that addition of that IL-7 and IL-15 and immediate stimulation of T cells post-nucleofection enhances cell viability.
• Figure 9 is a series of graphs showing that IL-7 and IL-15 rescue T cells from DNA mediated toxicity
• Figure 10 is a series of graphs showing that immediate stimulation of T cells post- nucleofection enhances cell viability.
• Figure 11 A-C is a series of graphs depicting T cell transposition with varying amounts of DNA.
• Primary human pan T cells were nucleofected with varying amounts of
• T cells were nucleofected with the indicated amounts of transposon and 5 ⁇ g sPBo mRNA. Cells were then stimulated on day 2 post-nucleofection through CD3 and CD28. As expected, T cells nucleofected with high amounts of DNA exhibited high episomal expression at day 1 post nucleofection whereas almost no episomal expression was observed at low DNA doses. In contrast, following expansion at day 21 post nucleofection the greatest percentage of transgene positive cells were observed in lower DNA amounts peaking at 1.67 ⁇ g for this transposon.
• A Flow analysis for transgene positive cells at day 1 and 21.
• B Percentage of transgene positive T cells.
• C Percentage of viable T cells at day 1 and 21. For all graphs shown in this figure, the Y-axis ranges from 0 to 100% in increments of 20% and the X-axis ranges from 0 to 10 5 by powers of 10.
• FIG. 12A-B is a series of graphs depicting T cell transposition with low DNA amounts using the Sleeping BeautyTM 100X (SB100X) transposase.
• Primary human pan T cells were nucleofected with GFP plasmids encoding either the piggyBacTM (PB) or Sleeping BeautyTM (SB) ITRs.
• PB piggyBacTM
• SB Sleeping BeautyTM
• A Cells were nucleofected with the indicated amounts of SB transposon and 1 ⁇ g SB transposase mRNA.
• B Cells were nucleofected with the indicated amounts of SB transposase and 0.75 ⁇ g SB transposon. Flow analysis was performed on day 14 post nucleofection for all samples. For all graphs shown in this figure, the Y-axis ranges from 0 to 250K in increments of 50K and the X-axis ranges from 0 to 10 5 by powers of 10.
• compositions and methods for the ex-vivo genetic modification of an immune cell comprising delivering to the immune cell, (a) a nucleic acid or amino acid sequence comprising a sequence encoding a transposase enzyme and (b) a recombinant and non-naturally occurring DNA sequence comprising a DNA sequence encoding a transposon.
• the method further comprises the step of stimulating the immune cell with one or more cytokine(s).
• Centyrins of the disclosure may comprise a protein scaffold, wherein the scaffold is capable of specifically binding an antigen.
• Centyrins of the disclosure may comprise a protein scaffold comprising a consensus sequence of at least one fibronectin type III (FN3) domain, wherein the scaffold is capable of specifically binding an antigen.
• the at least one fibronectin type III (FN3) domain may be derived from a human protein.
• the human protein may be Tenascin-C.
• the consensus sequence may comprise
• the consensus sequence may comprise an amino sequence at least 74% identical to
• the consensus sequence may encoded by a nucleic acid sequence comprising
• the consensus sequence may be modified at one or more positions within (a) a A-B loop comprising or consisting of the amino acid residues TEDS (SEQ ID NO: 7).
• the consensus sequence may be modified at one or more positions within (a) a A-B loop comprising or consisting of the amino acid residues TEDS (SEQ ID NO: 7).
• Centyrins of the disclosure may comprise a consensus sequence of at least 5 fibronectin type III (FN3) domains, at least 10 fibronectin type III (FN3) domains or at least 15 fibronectin type III
• the scaffold may bind an antigen with at least one affinity selected from a
• KD of less than or equal to 10 ⁇ 9 M, less than or equal to 10 ⁇ 10 M, less than or equal to 10 ⁇ n M, less than or equal to 10 ⁇ 12 M, less than or equal to 10 ⁇ 1 M, less than or equal to 10 ⁇ 14 M, and less than or equal to 10 ⁇ 15 M.
• the KD may be determined by surface plasmon resonance.
• antibody mimetic is intended to describe an organic compound that specifically binds a target sequence and has a structure distinct from a naturally-occurring antibody.
• Antibody mimetics may comprise a protein, a nucleic acid, or a small molecule.
• the target sequence to which an antibody mimetic of the disclosure specifically binds may be an antigen.
• Antibody mimetics may provide superior properties over antibodies including, but not limited to, superior solubility, tissue penetration, stability towards heat and enzymes (e.g. resistance to enzymatic degradation), and lower production costs.
• Exemplary antibody mimetics include, but are not limited to, an affibody, an afflilin, an affimer, an affitin, an alphabody, an anticalin, and avimer (also known as avidity multimer), a DARPin (Designed Ankyrin Repeat Protein), a Fynomer, a Kunitz domain peptide, and a monobody.
• Affibody molecules of the disclosure comprise a protein scaffold comprising or consisting of one or more alpha helix without any disulfide bridges.
• affibody molecules of the disclosure comprise or consist of three alpha helices.
• an affibody molecule of the disclosure may comprise an immunoglobulin binding domain.
• An affibody molecule of the disclosure may comprise the Z domain of protein A.
• Affilin molecules of the disclosure comprise a protein scaffold produced by modification of exposed amino acids of, for example, either gamma-B crystallin or ubiquitin. Affilin molecules functionally mimic an antibody's affinity to antigen, but do not structurally mimic an antibody. In any protein scaffold used to make an affilin, those amino acids that are accessible to solvent or possible binding partners in a properly-folded protein molecule are considered exposed amino acids. Any one or more of these exposed amino acids may be modified to specifically bind to a target sequence or antigen.
• Affimer molecules of the disclosure comprise a protein scaffold comprising a highly stable protein engineered to display peptide loops that provide a high affinity binding site for a specific target sequence.
• Exemplary affimer molecules of the disclosure comprise a protein scaffold based upon a cystatin protein or tertiary structure thereof.
• Exemplary affimer molecules of the disclosure may share a common tertiary structure of comprising an alpha- helix lying on top of an anti-parallel beta-sheet.
• Affitin molecules of the disclosure comprise an artificial protein scaffold, the structure of which may be derived, for example, from a DNA binding protein (e.g. the DNA binding protein Sac7d).
• Affitins of the disclosure selectively bind a target sequence, which may be the entirety or part of an antigen.
• exemplary affitins of the disclosure are
• Alphabody molecules of the disclosure may also be referred to as Cell-Penetrating Alphabodies (CPAB).
• CPAB Cell-Penetrating Alphabodies
• Alphabody molecules of the disclosure comprise small proteins (typically of less than 10 kDa) that bind to a variety of target sequences (including antigens).
• Alphabody molecules are capable of reaching and binding to intracellular target sequences.
• alphabody molecules of the disclosure comprise an artificial sequence forming single chain alpha helix (similar to naturally occurring coiled-coil structures).
• Alphabody molecules of the disclosure may comprise a protein scaffold comprising one or more amino acids that are modified to specifically bind target proteins. Regardless of the binding specificity of the molecule, alphabody molecules of the disclosure maintain correct folding and thermostability.
• Anticalin molecules of the disclosure comprise artificial proteins that bind to target sequences or sites in either proteins or small molecules.
• Anticalin molecules of the disclosure may comprise an artificial protein derived from a human lipocalin.
• Anticalin molecules of the disclosure may be used in place of, for example, monoclonal antibodies or fragments thereof.
• Anticalin molecules may demonstrate superior tissue penetration and thermostability than monoclonal antibodies or fragments thereof.
• Exemplary anticalin molecules of the disclosure may comprise about 180 amino acids, having a mass of approximately 20 kDa.
• anticalin molecules of the disclosure comprise a barrel structure comprising antiparallel beta-strands pairwise connected by loops and an attached alpha helix.
• anticalin molecules of the disclosure comprise a barrel structure comprising eight antiparallel beta-strands pairwise connected by loops and an attached alpha helix.
• Avimer molecules of the disclosure comprise an artificial protein that specifically binds to a target sequence (which may also be an antigen). Avimers of the disclosure may recognize multiple binding sites within the same target or within distinct targets. When an avimer of the disclosure recognize more than one target, the avimer mimics function of a bi- specific antibody.
• the artificial protein avimer may comprise two or more peptide sequences of approximately 30-35 amino acids each. These peptides may be connected via one or more linker peptides. Amino acid sequences of one or more of the peptides of the avimer may be derived from an A domain of a membrane receptor.
• Avimers have a rigid structure that may optionally comprise disulfide bonds and/or calcium. Avimers of the disclosure may demonstrate greater heat stability compared to an antibody.
• DARPins (Designed Ankyrin Repeat Proteins) of the disclosure comprise genetically-engineered, recombinant, or chimeric proteins having high specificity and high affinity for a target sequence.
• DARPins of the disclosure are derived from ankyrin proteins and, optionally, comprise at least three repeat motifs (also referred to as repetitive structural units) of the ankyrin protein.
• Ankyrin proteins mediate high-affinity protein-protein interactions.
• DARPins of the disclosure comprise a large target interaction surface.
• Fynomers of the disclosure comprise small binding proteins (about 7 kDa) derived from the human Fyn SH3 domain and engineered to bind to target sequences and molecules with equal affinity and equal specificity as an antibody.
• Kunitz domain peptides of the disclosure comprise a protein scaffold comprising a Kunitz domain.
• Kunitz domains comprise an active site for inhibiting protease activity.
• Structurally, Kunitz domains of the disclosure comprise a disulfide-rich alpha+beta fold. This structure is exemplified by the bovine pancreatic trypsin inhibitor.
• Kunitz domain peptides recognize specific protein structures and serve as competitive protease inhibitors.
• Kunitz domains of the disclosure may comprise Ecallantide (derived from a human lipoprotein- associated coagulation inhibitor (LACI)).
• LACI human lipoprotein- associated coagulation inhibitor
• Monobodies of the disclosure are small proteins (comprising about 94 amino acids and having a mass of about 10 kDa) comparable in size to a single chain antibody. These genetically engineered proteins specifically bind target sequences including antigens.
• Monobodies of the disclosure may specifically target one or more distinct proteins or target sequences.
• monobodies of the disclosure comprise a protein scaffold mimicking the structure of human fibronectin, and more preferably, mimicking the structure of the tenth extracellular type III domain of fibronectin.
• the tenth extracellular type III domain of fibronectin, as well as a monobody mimetic thereof, contains seven beta sheets forming a barrel and three exposed loops on each side corresponding to the three
• CDRs complementarity determining regions
• a monobody In contrast to the structure of the variable domain of an antibody, a monobody lacks any binding site for metal ions as well as a central disulfide bond. Multispecific monobodies may be optimized by modifying the loops BC and FG. Monobodies of the disclosure may comprise an adnectin.
• “about” can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1 % of a given value.
• the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
• the disclosure provides isolated or substantially purified polynucleotide or protein compositions.
• An “isolated” or “purified” polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment.
• an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
• an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
• isolated polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived.
• the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived.
• a protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1 % (by dry weight) of contaminating protein.
• optimally culture medium represents less than about 30%, 20%,
• fragment refers to a portion of the DNA sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a DNA sequence comprising coding sequences may encode protein fragments that retain biological activity of the native protein and hence DNA recognition or binding activity to a target DNA sequence as herein described. Alternatively, fragments of a DNA sequence that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a DNA sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the invention.
• Nucleic acids or proteins of the disclosure can be constructed by a modular approach including preassembling monomer units and/or repeat units in target vectors that can subsequently be assembled into a final destination vector.
• Polypeptides of the disclosure may comprise repeat monomers of the disclosure and can be constructed by a modular approach by preassembling repeat units in target vectors that can subsequently be assembled into a final destination vector.
• the disclosure provides polypeptide produced by this method as well nucleic acid sequences encoding these polypeptides.
• the disclosure provides host organisms and cells comprising nucleic acid sequences encoding polypeptides produced this modular approach.
• antibody is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with polyepitopic specificity. It is also within the scope hereof to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as "analogs") of the antibodies hereof as defined herein. Thus, according to one embodiment hereof, the term “antibody hereof in its broadest sense also covers such analogs. Generally, in such analogs, one or more amino acid residues may have been replaced, deleted and/or added, compared to the antibodies hereof as defined herein.
• Antibody fragment and all grammatical variants thereof, as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein the portion is free of the constant heavy chain domains (i.e. CH2,
• antibody fragments include Fab, Fab', Fab'- SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a
• single-chain antibody fragment or “single chain polypeptide”), including without limitation
• single-chain Fv (scFv) molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific or multivalent structures formed from antibody fragments.
• the heavy chain(s) can contain any constant domain sequence (e.g.
• CHI in the IgG isotype found in a non-Fc region of an intact antibody, and/or can contain any hinge region sequence found in an intact antibody, and/or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain(s).
• the term further includes single domain antibodies ("sdAB") which generally refers to an antibody fragment having a single monomeric variable antibody domain, (for example, from camelids). Such antibody fragment types will be readily understood by a person having ordinary skill in the art.
• Binding refers to a sequence-specific, non-covalent interaction between
• macromolecules e.g., between a protein and a nucleic acid. Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific.
• compositions and methods include the recited elements, but do not exclude others.
• Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants or inert carriers.
• Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.
• epitope refers to an antigenic determinant of a polypeptide.
• An epitope could comprise three amino acids in a spatial conformation, which is unique to the epitope.
• an epitope consists of at least 4, 5, 6, or 7 such amino acids, and more usually, consists of at least 8, 9, or 10 such amino acids.
• Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.
• expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
• 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, shRNA, micro RNA, 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.
• Modulation or “regulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.
• operatively linked or its equivalents (e.g., “linked operatively") means two or more molecules are positioned with respect to each other such that they are capable of interacting to affect a function attributable to one or both molecules or a combination thereof.
• Non-covalently linked components and methods of making and using non-covalently linked components are disclosed.
• the various components may take a variety of different forms as described herein.
• non-covalently linked (i.e., operatively linked) proteins may be used to allow temporary interactions that avoid one or more problems in the art.
• the ability of non-covalently linked components, such as proteins, to associate and dissociate enables a functional association only or primarily under circumstances where such association is needed for the desired activity.
• the linkage may be of duration sufficient to allow the desired effect.
• a method for directing proteins to a specific locus in a genome of an organism is disclosed.
• the method may comprise the steps of providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule are capable of operatively linking via a non-covalent linkage.
• scFv refers to a single-chain variable fragment.
• scFv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a linker peptide.
• the linker peptide may be from about 5 to 40 amino acids or from about 10 to 30 amino acids or about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length.
• Single-chain variable fragments lack the constant Fc region found in complete antibody molecules, and, thus, the common binding sites (e.g., Protein G) used to purify antibodies.
• the term further includes a scFv that is an intrabody, an antibody that is stable in the cytoplasm of the cell, and which may bind to an intracellular protein.
• single domain antibody means an antibody fragment having a single monomeric variable antibody domain which is able to bind selectively to a specific antigen.
• a single-domain antibody generally is a peptide chain of about 110 amino acids long, comprising one variable domain (VH) of a heavy-chain antibody, or of a common IgG, which generally have similar affinity to antigens as whole antibodies, but are more heat-resistant and stable towards detergents and high concentrations of urea. Examples are those derived from camelid or fish antibodies.
• single-domain antibodies can be made from common murine or human IgG with four chains.
• a specific binding interaction will discriminate between desirable and undesirable antigens in a sample, in some embodiments more than about ten- to 100-fold or more (e.g., more than about 1000- or 10,000-fold).
• “Specificity” refers to the ability of an immunoglobulin or an immunoglobulin fragment, such as a nanobody, to bind preferentially to one antigenic target versus a different antigenic target and does not necessarily imply high affinity.
• a "target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.
• nucleic acid or “oligonucleotide” or “polynucleotide” refer to at least two nucleotides covalently linked together.
• the depiction of a single strand also defines the sequence of the complementary strand.
• a nucleic acid may also encompass the complementary strand of a depicted single strand.
• a nucleic acid of the disclosure also encompasses substantially identical nucleic acids and complements thereof that retain the same structure or encode for the same protein.
• Probes of the disclosure may comprise a single stranded nucleic acid that can hybridize to a target sequence under stringent hybridization conditions.
• nucleic acids of the disclosure may refer to a probe that hybridizes under stringent hybridization conditions.
• Nucleic acids of the disclosure may be single- or double-stranded. Nucleic acids of the disclosure may contain double-stranded sequences even when the majority of the molecule is single-stranded. Nucleic acids of the disclosure may contain single-stranded sequences even when the majority of the molecule is double-stranded. Nucleic acids of the disclosure may include genomic DNA, cDNA, RNA, or a hybrid thereof. Nucleic acids of the disclosure may contain combinations of deoxyribo- and ribo-nucleotides.
• Nucleic acids of the disclosure may contain combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids of the disclosure may be synthesized to comprise non-natural amino acid modifications. Nucleic acids of the disclosure may be obtained by chemical synthesis methods or by recombinant methods.
• Nucleic acids of the disclosure may be non-naturally occurring. Nucleic acids of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring. Nucleic acids of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring. Nucleic acids of the disclosure may contain modified, artificial, or synthetic nucleotides that do not naturally-occur, rendering the entire nucleic acid sequence non- naturally occurring.
• nucleotide sequences may encode any particular protein. All such nucleotides sequences are contemplated herein.
• operably linked refers to the expression of a gene that is under the control of a promoter with which it is spatially connected.
• a promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under its control.
• the distance between a promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. Variation in the distance between a promoter and a gene can be accommodated without loss of promoter function.
• promoter refers to a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell.
• a promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same.
• a promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
• a promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
• a promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.
• promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1 Alpha promoter, CAG promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.
• the term "substantially complementary” refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.
• the term "substantially identical” refers to a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.
• nucleic acid refers to (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.
• vector refers to a nucleic acid sequence containing an origin of replication.
• a vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
• a vector can be a DNA or RNA vector.
• a vector can be a self-replicating extrachromosomal vector, and preferably, is a DNA plasmid.
• a vector may comprise a combination of an amino acid with a DNA sequence, an RNA sequence, or both a DNA and an RNA sequence.
• variant when used to describe a peptide or polypeptide, refers to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity.
• Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity.
• a conservative substitution of an amino acid i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al, J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. Amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ⁇ 2 are substituted.
• hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function.
• a consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity.
• U.S. Patent No. 4,554,101 incorporated fully herein by reference.
• substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity. Substitutions can be performed with amino acids having hydrophilicity values within ⁇ 2 of each other. Both the
• hyrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
• fusion polypeptides and/or nucleic acids encoding such fusion polypeptides include conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table A.
• conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-77) as set forth in Table B.
• polypeptides of the disclosure are intended to include polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues.
• Polypeptides or nucleic acids of the disclosure may contain one or more conservative substitution.
• the term "more than one" of the aforementioned amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the recited amino acid substitutions.
• the term “more than one” may refer to 2, 3, 4, or 5 of the recited amino acid substitutions.
• Polypeptides and proteins of the disclosure may be non-naturally occurring.
• Polypeptides and proteins of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire amino acid sequence non-naturally occurring.
• Polypeptides and proteins of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally-occur, rendering the entire amino acid sequence non-naturally occurring.
• Polypeptides and proteins of the disclosure may contain modified, artificial, or synthetic amino acids that do not naturally- occur, rendering the entire amino acid sequence non-naturally occurring.
• sequence identity may be determined by using the stand-alone executable BLAST engine program for blasting two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety).
• NCBI National Center for Biotechnology Information
• identity when used in the context of two or more nucleic acids or polypeptide sequences, refer to a specified percentage of residues that are the same over a specified region of each of the sequences.
• the percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue 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 specified region, and multiplying the result by 100 to yield the percentage of sequence identity.
• the residues of single sequence are included in the denominator but not the numerator of the calculation.
• thymine (T) and uracil (U) can be considered equivalent.
• Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.
• endogenous refers to nucleic acid or protein sequence naturally associated with a target gene or a host cell into which it is introduced.
• exogenous refers to nucleic acid or protein sequence not naturally associated with a target gene or a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., DNA sequence, or naturally occurring nucleic acid sequence located in a non- naturally occurring genome location.
• the disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell. By “introducing” is intended presenting to the plant the polynucleotide construct in such a manner that the construct gains access to the interior of the host cell.
• the methods of the invention do not depend on a particular method for introducing a polynucleotide construct into a host cell, only that the polynucleotide construct gains access to the interior of one cell of the host.
• Methods for introducing polynucleotide constructs into bacteria, plants, fungi and animals are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
• stable transformation is intended that the polynucleotide construct introduced into a plant integrates into the genome of the host and is capable of being inherited by progeny thereof.
• transient transformation is intended that a polynucleotide construct introduced into the host does not integrate into the genome of the host.
• the term "genetically modified plant (or transgenic plant)” refers to a plant which comprises within its genome an exogenous polynucleotide.
• the exogenous polynucleotide is stably integrated into the genome such that the polynucleotide is passed on to successive generations.
• the exogenous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
• Transgenic is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of exogenous nucleic acid including those trans genies initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic.
• the term "transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra- chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
• the term "modifying" is intended to mean that the sequence is considered modified simply by the binding of the polypeptide. It is not intended to suggest that the sequence of nucleotides is changed, although such changes (and others) could ensue following binding of the polypeptide to the nucleic acid of interest.
• the nucleic acid sequence is DNA. Modification of the nucleic acid of interest (in the sense of binding thereto by a polypeptide modified to contain modular repeat units) could be detected in any of a number of methods (e.g. gel mobility shift assays, use of labelled polypeptides—labels could include radioactive, fluorescent, enzyme or
• biotin/streptavidin labels Modification of the nucleic acid sequence of interest (and detection thereof) may be all that is required (e.g. in diagnosis of disease). Desirably, however, further processing of the sample is performed. Conveniently the polypeptide (and nucleic acid sequences specifically bound thereto) is separated from the rest of the sample.
• the polypeptide-DNA complex is bound to a solid phase support, to facilitate such separation.
• the polypeptide may be present in an acrylamide or agarose gel matrix or, more preferably, is immobilized on the surface of a membrane or in the wells of a microtitre plate.
• Every maximum numerical limitation given throughout this disclosure includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this disclosure will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this disclosure will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
• the piggyBacTM (PB) transposon system was used for genetically modifying human lymphocytes for production of autologous CAR-T immunotherapies and other applications.
• T Lymphocytes purified from patient blood or apheresis product was electroporated with a plasmid DNA transposon and a transposase.
• electroporation systems include the Neon (Thermo Fisher), BTX ECM 830 (Harvard Apparatus), Gene Pulser (BioRad), MaxCyte PulseAgile
• the first plasmid was a pmaxGFPTM plasmid that is provided as a control plasmid in the Lonza Amaxa nucleofection kit. It is highly purified by HPLC and does not contain endotoxin at detectable levels.
• the second plasmid was our in-house produced PB transposon encoding a human EF1 alpha promoter driving GFP. Transfection efficiency, as measured by GFP expression from the electroporated plasmid, and cell viability was assessed by FACS at days 2, 3, and 6 post-electroporation.
• the cells exhibited a 2.0-fold reduction in cell viability at day 1 and a 13.2-fold reduction at day 4.
• sPBo Super piggyBacTM
• the current plasmid transposon also contains a backbone required for plasmid amplification in bacteria, it is possible to significantly reduce the total amount of DNA by excluding this sequence. This may be achieved by restriction digest of the plasmid transposon prior to the nucleofection reaction. In addition, this could be achieved by administering the transposon as a PCR product or as a doggyboneTM DNA, which is a double stranded DNA that is produced in vitro by a mechanism that excludes the initial backbone elements required for bacterial replication of the plasmid.
• plasmid DNA is toxic in primary T lymphocytes, but we have observed that this toxic effect is not as dramatic in tumor cell lines and other transformed cells. Based upon this observation, we hypothesized that primary T lymphocytes may be refractory to plasmid DNA transfection due to heightened DNA sensing pathways, which would protect immune cells from infection by viruses and bacteria. If these data are a result of heightened DNA sensing mechanisms, then it may be possible to enhance plasmid transfection efficiency and/or cell viability by the addition of DNA sensing pathway inhibitors to the post-nucleofection reaction.
• reagents that inhibited the TLR-9 pathway, caspase pathway, or those involved in cytoplasmic double stranded DNA sensing.
• These reagents include Bafilomycin Al, which is an autophagy inhibitor that interferes with endosomal acidification and blocks NFkB signaling by TLR9, Chloroquine, which is a TLR9 antagonist, Quinacrine, which is a TLR9 antagonist and a cGAS antagonist, AC-YVAD-CMK, which is a caspase 1 inhibitor targeting the AIM2 pathway, Z-VAD-FMK, which is a pan caspase inhibitor, Z-IETD-FMK, which is a caspase 8 inhibitor triggered by the TLR9 pathway.
• Bafilomycin Al which is an autophagy inhibitor that interferes with endosomal acidification and blocks NFkB signaling by TLR9
• Chloroquine which is a TLR9 antagonist
• Quinacrine which is a TLR9 antagonist and a
• IL7 and IL15 were added either 1 hour or 1 day following electroporation, enhanced viability over 3 -fold when compared with introduction of the plasmid transposon alone without additional treatment.
• stimulation of the T cells post-nucleofection using either activator or expander beads also dramatically enhanced T cell viability; stimulation was better when the beads were added 1 hour or 1 day post-nucleofection as compared to adding them 2 days post.
• ROCK inhibitor ROCK inhibitor and the removal of dead cells from the culture using the Dead Cell Removal kit from Miltenyi, but saw no improvement in cell viability.

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• 2017
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