WO2022266467A2 - Recombinant histone polypeptide and uses thereof - Google Patents

Recombinant histone polypeptide and uses thereof Download PDF

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Publication number
WO2022266467A2
WO2022266467A2 PCT/US2022/034035 US2022034035W WO2022266467A2 WO 2022266467 A2 WO2022266467 A2 WO 2022266467A2 US 2022034035 W US2022034035 W US 2022034035W WO 2022266467 A2 WO2022266467 A2 WO 2022266467A2
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cell
histone
polypeptide
seq
cells
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PCT/US2022/034035
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French (fr)
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WO2022266467A3 (en
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William G. Kaelin Jr.
Parker SULKOWSKI
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Dana-Farber Cancer Institute, Inc.
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Publication of WO2022266467A2 publication Critical patent/WO2022266467A2/en
Publication of WO2022266467A3 publication Critical patent/WO2022266467A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • This invention is directed to isolated or recombinant histone polypeptides and fragments thereof, compositions comprising the same, and methods for using the same to deliver macromolecules to a cell.
  • a histone is a protein that provides structural support to a chromosome. In order for very long DNA molecules to fit into the cell nucleus, they wrap around complexes of histone proteins, giving the chromosome a more compact shape. Some variants of histones are associated with the regulation of gene expression.
  • An aspect of the invention is directed to an isolated or recombinant histone polypeptide comprising a peptide or fragment thereof having at least 90% identity to the amino acid sequence according to:
  • the isolated or recombinant histone polypeptide comprises or consists of the amino acid sequence according to SEQ ID NO: 1, 2, or 3.
  • the isolated or recombinant histone polypeptide comprises one or more modifications.
  • the one or more modifications comprises one or more post- translational modifications and/or mutations.
  • the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
  • the mutation one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
  • the isolated or recombinant histone polypeptide fragment comprises an N-terminus truncated fragment or a C-terminus truncated fragment.
  • the fragment consists of or comprises a fragment of SEQ ID NO: 1, 2 or 3, or a sequence at least 90% identical thereto.
  • the isolated or recombinant histone polypeptide exhibits at least one activity selected from the group consisting of crossing a cell membrane, secretion from a cell, uptake by a cell, or delivery of a macromolecule.
  • the isolated or recombinant histone polypeptide is a eukaryotic histone polypeptide.
  • the isolated or recombinant histone polypeptide is a mammalian histone polypeptide.
  • the isolated or recombinant histone polypeptide is isolated from a host cell which expresses the polypeptide.
  • the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell.
  • the isolated or recombinant histone polypeptide is encoded by a nucleic acid sequence according to 4, a fragment thereof, or a sequence at least 90% identical thereto.
  • the isolated or recombinant histone polypeptide comprises a fusion protein.
  • the fusion protein comprises at least one functional moiety.
  • the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof.
  • the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin.
  • the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
  • the functional moiety comprises a macromolecule.
  • the macromolecule comprises a protein (such as an antibody, a cytokine, or a growth-inhibitor) or a chemical compound (such as a chemotherapeutic or toxin).
  • aspects of the invention are also drawn to a nucleic acid encoding a histone polypeptide or fragment thereof according to SEQ ID NO: 1, 2, or 3, or an amino acid sequence having at least 90% identity thereto.
  • the nucleic acid comprises or consists of a sequence having at least 90% identity to SEQ ID NO: 4 or a fragment thereof. In embodiments the nucleic acid comprises or consists of SEQ ID NO: 4.
  • aspects of the invention are also drawn to a cell comprising a nucleic acid encoding an isolated or recombinant histone polypeptide thereof.
  • the cell is a mammalian cell, bacterial cell, yeast cell, or an insect cell.
  • aspects of the invention are drawn to a fusion protein comprising an isolated or recombinant histone polypeptide comprising or consisting of an amino acid sequence of at least 90% identity to SEQ ID NO: 1, 2, or 3 or a fragment thereof and at least one functional moiety.
  • the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof.
  • the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin.
  • the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
  • the functional moiety comprises a macromolecule.
  • the macromolecule comprises a protein (such as an antibody, a cytokine, or a growth-inhibitor) or a chemical compound (such as a chemotherapeutic or a toxin).
  • a protein such as an antibody, a cytokine, or a growth-inhibitor
  • a chemical compound such as a chemotherapeutic or a toxin
  • the histone polypeptide comprises or consists of SEQ ID NO: 1, 2, or 3.
  • the histone polypeptide comprises one or more modifications.
  • the one or more modifications comprises one or more post-translational modifications and/or mutations.
  • the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
  • the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
  • aspects of the invention are also drawn to a cell expressing an isolated or recombinant histone polypeptide thereof, or the fusion protein comprising or consisting of an isolated or recombinant histone polypeptide thereof.
  • the cell comprises an insect cell, a mammalian cell, a bacterial, or a yeast cell.
  • aspects of the invention are drawn to a composition comprising an isolated or recombinant histone polypeptide thereof, a fusion protein comprising or consisting of an isolated or recombinant histone polypeptide thereof, or a cell comprising the same, and a pharmaceutically acceptable carrier.
  • composition further comprises at least one additional active agent.
  • additional active agent e.g., a drug-delivery platform comprising an isolated or recombinant histone polypeptide comprising or consisting of an amino acid sequence of at least 90% identity to SEQ ID NO: 1, 2, or 3 or a fragment thereof.
  • the drug deliver platform comprises an isolated or recombinant histone polypeptide thereof comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, 2 or 3.
  • the isolated or recombinant histone polypeptide thereof of the drug- delivery platform comprises one or more modifications.
  • the one or more modifications comprises one or more post-translational modifications and/or mutations.
  • the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
  • the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
  • the drug-delivery platform comprises a fusion protein comprising an isolated or recombinant histone polypeptide thereof.
  • the fusion protein comprises at least one functional moiety.
  • the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof.
  • the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin.
  • the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
  • the functional moiety comprises a macromolecule.
  • the macromolecule comprises a protein (such as an antibody, a cytokine, or a growth-factor) or chemical compound (such as a chemotherapeutic or toxin).
  • a protein such as an antibody, a cytokine, or a growth-factor
  • chemical compound such as a chemotherapeutic or toxin.
  • Aspects of the invention are further drawn to a method of delivering at least one functional moiety to a cell.
  • the cell can be a cancer cell, or the cell can be a non-cancer cell.
  • the method comprises contacting a cell with an isolated or recombinant histone polypeptide comprising the amino acid sequence of at least 90% identity to SEQ ID NO: 1, 2, or 3, or a fragment thereof, wherein the at least one functional moiety is fused to the histone polypeptide.
  • the isolated or recombinant histone polypeptide thereof comprises or consists of SEQ ID NO: 1, 2 or 3.
  • the histone polypeptide comprises one or more modifications.
  • the one or more modifications comprises one or more post-translational modifications and/or mutations.
  • the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
  • the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
  • the functional moiety can be a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof.
  • the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin.
  • the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
  • the functional moiety comprises a macromolecule.
  • the macromolecule comprises a protein (such as an antibody, a cytokine, or a growth inhibitor) or chemical compound (such as a chemotherapeutic or toxin).
  • aspects of the invention are also drawn towards a method of treating a subject afflicted with a disease, such as cancer.
  • the method comprises administering to the subject an isolated or recombinant histone polypeptide comprising an amino acid sequence of at least 90% identity to SEQ ID NO: 1, 2, or 3 or a fragment thereof, wherein the histone polypeptide is fused to a functional moiety.
  • the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof.
  • the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin.
  • the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
  • the functional moiety comprises a macromolecule.
  • the macromolecule comprises a protein (such as an antibody, a cytokine, or a growth inhibitor) or chemical compound (such as a chemotherapeutic or toxin).
  • the isolated or recombinant histone polypeptide thereof comprises or consists of SEQ ID NO: 1, 2, or 3.
  • the isolated or recombinant histone polypeptide thereof comprises one or more modifications.
  • the one or more modifications comprises one or more post-translational modifications and/or mutations.
  • the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
  • the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
  • Embodiments can further comprise administering to the subject an isolated or recombinant histone polypeptide fused to a functional moiety.
  • the isolated or recombinant histone polypeptide comprises histone H3.
  • histone H3 comprises histone H3.1, histone H3.2, or histone H3.3.
  • the isolated or recombinant histone polypeptide comprises or consists of an amino acid sequence or fragment thereof according to SEQ ID NO: 1, 2 or 3, or a sequence at least 90% identical thereto.
  • the isolated or recombinant histone polypeptide comprises one or more modifications.
  • the one or more modifications comprises one or more post- translational modifications and/or mutations.
  • the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
  • the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
  • the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof.
  • the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin.
  • the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
  • the functional moiety comprises a macromolecule.
  • the macromolecule comprises a protein (such as an antibody, a cytokine, or growth inhibitor) or chemical compound (such as a chemotherapeutic or toxin).
  • aspects of the invention are also drawn towards a method of transducing a cell with a protein or chemical compound.
  • the method comprises contacting the cell with an isolated or recombinant histone polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1, 2, or 3 or a fragment thereof, wherein the histone polypeptide is fused to the protein or chemical compound.
  • the cell can be a cancer cell, or the cell can be a non-cancer cell.
  • the histone polypeptide comprises or consists of SEQ ID NO: 1, 2 or
  • the histone polypeptide comprises one or more modifications.
  • the one or more modifications comprises one or more post-translational modifications and/or mutations.
  • the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
  • the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
  • the protein comprises a therapeutic moiety or an imaging moiety.
  • the therapeutic moiety comprises a therapeutic protein (such as an antibody, a cytokine, or a growth inhibitor) or polypeptide, a small molecule (such as a chemical compound or chemotherapeutic), or a toxin.
  • the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
  • FIG. 1 shows BirA is an E. coli biotin ligase that covalently attaches biotin to nearby proteins.
  • FIG. 2 shows methods of identifying secreted proteins using ER-targeted BirA.
  • FIG. 3 shows 786-0 VHL Renal Cancer Cells Expressing ER-BirA and Dox- inducible VHL.
  • FIG. 4 shows detection of biotinylated secreted proteins.
  • FIG. 5 shows detection of extracellular H3 without the BirA system.
  • FIG. 6 shows posttranslational modifications on secreted H3 do not mirror bulk H3.
  • FIG. 7 shows H3K14 acetylation is highly enriched in secreted H3.
  • FIG. 8 shows secretion of wildtype H3 and H3K14 mutants. The mutation of K to
  • R blocks acetylation, and the mutation of K to Q mimics acetylation.
  • FIG. 9A shows secretion of H3 is autophagy-dependent.
  • Brefeldin A blocks canonical protein secretion
  • Bafilomycin A1 blocks autophagy-dependent secretion.
  • FIG. 9B shows secretion of H3 is autophagy-dependent using ATG7 knockout.
  • FIG. 9C shows secretion of H3 is autophagy-dependent using Beclin knockout.
  • FIG. 10 shows serum starvation promotes H3 secretion.
  • FIG. 11 shows autophagy inducer Rapamycin promotes H3 secretion.
  • FIG. 12 shows Dox-inducible H3 and H4 mEmerald and mCherry fusion proteins.
  • FIG. 13 shows detection of dual H3-mEmerald and H3-mCherry positive cells in co-culture experiments.
  • FIG. 14 shows co-culture H3-cherry 293T cells with Myr (membrane)-GFP RCC4 cells resulting in rare green cells (myr-GFP) with red nuclear inclusions (H3-mcherry).
  • FIG. 15 shows inclusion corresponds to condensed chromosomes at metaphase plate.
  • FIG. 16 shows an experimental design of a transwell system. Pore size can be, for example, 0.2mM. Results show horizontal transfer of histone H3.
  • FIG. 17 shows “stamp upon receipt assay”, which demonstrates evidence of Histone H3 transfer using biotinylated H3-FLAG. Results show detection of H3 in the nucleus of recipient cells.
  • FIG. 18 shows single molecule imaging using HaloTag-H3 (or H4) 293T cells.
  • media is harvested from HaloTag-H3 (or H4) 293T cells and then added to mCherry RCC4 (Renal Cell Carcinoma) cells.
  • Results show starvation selectively promotes H3 secretion into the culture medium, and also imaging uptake of Halo-Tag-Histone H3 into RCC cells. After incubation and transferring, 35 out of 217 RCC cells (16.1%) are positive for HaloTag signal.
  • FIG. 19 shows most of the H3 taken up by cells remains in the cytoplasm, but some gets to the nucleus.
  • FIG. 20 shows co-culture of rare HaloTag-Histone H3 293T donor cells and H4- mCherry RCC4 recipient cells.
  • FIG. 21 shows H3 transferred to nucleus and cytoplasm of recipient cells. Localization colored based on frame numbers.
  • FIG. 22 shows co-culture of Lox-Stop-Lox GFP reporter cells with H3-Cre expressing cells activates GFP expression.
  • FIG. 23 shows autophagy is not required for uptake of H3 (mCherry data).
  • FIG. 24 shows 50/50 tumors FACS data.
  • FIG. 25 shows co-culture with H3-Cre donor cells induces “color switch” recombination in recipient cells.
  • FIG. 26 shows “color switch” assay (Cre Reporter #1). See also, Zomeer et al. Nature Protocols, 2016.
  • FIG. 27 shows Histone H3-Cre fusion proteins induces “color switch” recombination in independent cells.
  • FIG. 29 shows GFP fluorescence in the “color switch” reporter assay is Cre- dependent.
  • FIG. 30 shows autophagy is not required for uptake of H3 (mEmerald data)
  • FIG. 31 shows glycine substitution (removing AA side chain) at K4, K9, K27, or K36 does not decrease the levels of tagged-H3 in conditioned media.
  • FIG. 32 shows K to Q (acetylation mimic) at K27 and K36 decreases accumulation of the tagged-H3 in conditioned media.
  • FIG. 33 shows dectection of BirA-dependent, biotinylated extracellular proteins.
  • FIG. 34 shows BirA-G3-ER system detects known, VHL-dependent differences in secreted peptides.
  • FIG. 35 shows no H3 is detected in unconditioned, concentrated media.
  • FIG. 36 shows detection of H3 secretion using orthogonal methodology (TCA precipitation) in multiple RCC cell lines using independent antibodies.
  • Cells 10pg of whole cell lysate;
  • Media TCA precipitated proteins from lmL serum-free OPTIMEM media after 48 hours in culture.
  • IGFBP3 is known not to be secreted by 769P cells.
  • FIG. 37 shows H3 and H4 fluorescent fusion proteins have similar fluorescence intensity.
  • FIG. 38 shows 293FT H3-mCherry and RCC4 myr-GFP co-culture assay: single color controls.
  • FIG. 39 shows 293FT H3-mCherry and RCC4 myr-GFP co-culture shows some “dual positive” cells.
  • FIG. 40 shows forward and side scatter dot plots show cell-type enriched populations in RCC4/293FT co-culture assays.
  • FIG. 41 shows “dual positive” cells only appear in the RCC4-enriched population.
  • FIG. 42 shows increasing the ratio of 293mCherry expressing cells increases the proportion of “dual positive” RCC4 cells.
  • FIG. 43 shows secreted H3 appears monomeric on native cells.
  • FIG. 44 shows annotated nucleic acid and amino acid sequences of histone H3 fused to Cre recombinase.
  • FIG. 45 shows annotated nucleic acid and amino acid sequences of histone H3 fused to mEmerald.
  • FIG. 46 shows annotated nucleic acid and amino acid sequences of histone H3 fused to mCherry.
  • FIG. 47 shows (a) schema for protein biotinylation by BirA.
  • b-e 786-0 VHL-/- renal carcinoma cells expressing BirA-ER or the corresponding empty vector (EV) were grown, where indicated, in the presence of 2 pg/mL doxy cy cline (Dox) for 4 days to induce exogenous VHL expression prior to the addition of 50 mM biotin.
  • Cell lysates (Cells) and secreted proteins captured on streptavidin beads (Media) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with horseradish peroxidase-conjugated streptavidin (b) or with the indicated antibodies (c-e).
  • FIG. 48 shows (a, f and g) immunoblot analysis of RCC4 cell extract and TCA precipitated conditioned media after the indicated treatments for 24 h.
  • Cells whole cell extracts.
  • Media TCA precipitated conditioned media
  • Cells whole cell extracts.
  • Media TCA precipitated conditioned media.
  • FIG. 49 shows (a and b). Data (a) and quantification (b) of 2 color FACS after co culturing 786-0 cells expressing the indicated histone fused to either mEmerald or mCherry (1 : 1 mix) in serum-free Opti-MEM for 2 days (c and d). Representative live cell fluorescence micrographs (c) and quantification (d) after coculturing 293T embryonic kidney cells expressing either H3 -mCherry or H4-mCherry with RCC4 cells expressing Myr-GFP in serum-free media for 2 days (e and 1).
  • FIG. 50 shows (a and e). Schemas for in vivo transfer experiments in tumor bearing nude mice. Data (b) and quantification (c) of 2 color FACS of dissociated tumors formed 5 weeks after implanting 786-0 cells expressing either H3-Cre or H4 Cre mixed 1 : 1 with RCC4 color switch reporter cells (d) Immunoblot analysis of tumors from (a-c).
  • 786-0 color switch reporter cells infected ex vivo with a Cre expressing lentivirus (+Cre) or the empty vector (EV) were included as controls (f and g)
  • 786-0 color switch reporter cells infected ex vivo with a Cre expressing lentivirus (+Cre) or the empty vector (EV) were included as controls (i-j) Immunoblot analysis of liver lysates (i) and serum (j) from c57/B16 mice that were or were not fasted for 24 h. Each lane contains the same from a different mouse. For c and g, each dot represents an independent tumor, bars are mean ⁇ s.e.m.
  • FIG. 51 shows (a) immunoblot analysis of cell extract from 786-0 cells expressing a doxycycline-inducible VHL. (b) 786-0 VEIL-/- renal carcinoma cells expressing BirA-ER or the corresponding empty vector (EV) were grown, where indicated, in the presence of doxycycline (Dox) for 4 days to induce exogenous VHL expression prior to the addition of 50 mM biotin.
  • Dox doxycycline
  • Cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with horseradish peroxidase-conjugated streptavidin or immunoblotted with the indicated antibodies
  • c Immunoblot analysis of 786-0 cell extracts (Cells) and increasing amounts of 786-0 cell conditioned or unconditioned media after concentration with a spin- column.
  • d Immunoblot analysis of 786-0, RCC4, UMRC2, 769P, and A498 cell extracts and TCA-precipitated conditioned media
  • e Immunoblot analysis of 293T, U20S, B16-F10, HT1080, MDA-MB-231, and MCF7 cell extracts and TCA-precipitated conditioned media.
  • FIG. 52 shows (a, b and c) data (a) and quantification (b,c) of FACS analysis of Propidium Iodide uptake of 786-0 cells that had been grown in Opti-MEM, Opti-MEM + 1% FBS, or Opti-MEM + indicated dose of Puromycin for 48 hours (d and e) Data (d) and quantification (e) of 2 color FACS analysis of Propidium Iodide uptake and Annexin V staining of 786-0 cells that had been grown in Opti-MEM, Opti-MEM + 1% FBS, or Opti- MEM + indicated dose of Puromycin for 48 hours.
  • Cell lysates (Cells) and secreted proteins captured on streptavidin beads (Media) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with horseradish peroxidase-conjugated streptavidin or with the indicated antibodies (j) Immunoblot analysis of TCA precipitated 786-0 conditioned media after the indicated treatments for 24 h.
  • FIG. 53 shows (a) representative live-cell fluorescence micrographs of 786-0 cells constitutively expressing H2A, H2B, H3, or H4 fused to either mEmerald or mCherry. (b) Representative live cell fluorescence micrographs of 786-0 cells expressing doxycycline- inducible H3 or H4 fused to either mEmerald or mCherry that were grown in media that did (+Dox) or did not (-Dox) contain lpg/mL Doxy cy cline for 4 days (c and d) Data (c) and quantification (d) of 2 color FACS after coculturing 786-0 cells expressing the indicated doxycycline-inducible H3 or H4 fused to either mEmerald or mCherry (1 : 1 mix) in serum- free Opti-MEM that did or did not contain 1 pg/mL doxy cy cline for 2 days (e and
  • FIG. 54 shows (a and b) data (a) and quantification (b) of 2 color FACS after co culturing RCC4 cells expressing the indicated histone fused to either mEmerald or mCherry (1:1 mix) in serum-free Opti-MEM or Opti-MEM+ 1% FBS at the indicated oxygen concentration for 2 days.
  • FIG. 55 shows (a and b) data (a) and quantification (b) of FACS analysis of recipient RCC4 cells that had first undergone CRISPR/Cas9 editing with indicated guide RNAs and then were grown in conditioned medium from 293T cells expressing either H3 or H4 fused to mEmerald for 48 hours (c and d) Data (c) and quantification (d) of FACS analysis of recipient RCC4 cells that had first undergone CRISPR/Cas9 editing with indicated guide RNAs, after 48h in culture with conditioned medium from 293T cells expressing either H3 or H4 fused to mCherry.
  • TIRF Total Internal Reflection fluorescence
  • top and bottom error bars represent 95 th and 5 th percentiles, respectively; box represents the range from the 25 th to 75 th percentile; center line represents the median and the small square represents mean value (***, p value ⁇ 0.01, paired sample T test) (c-d) HILO single molecule localization microscopy of H3 -Halo tag molecules transferred into recipient RCC4 cells after 24 h culture in conditioned Opti-MEM from H3-HaloTag expressing 293T cells.
  • the reconstructed images consist of localizations color coded by frame numbers (c) and local densities (d).
  • the nucleus is highlighted by the dotted circle. Scale bar, 10 pm.
  • FIG. 57 shows (a) immunoblot analysis of cell extracts and 3 mL of TCA precipitated conditioned media from 786-0 cells expressing the indicated H3 point mutants fused to Cre Recombinase (Cre) after 48 h culture in serum-free Opti-MEM.
  • FIG. 58 shows (a) representative live cell fluorescence micrographs of 786-0 cells expressing the indicated H3 point mutants fused to either mEmerald or mCherry. (b and c) Data (b) and quantification (c) of FACS after coculturing 786-0 cells expressing the indicated H3 point mutants fused to mCherry with RCC4 cells expressing myr-GFP (1:1 mix) in serum-free Opti-MEM + lpg/mL doxy cy cline for 2 days (d and e).
  • FIG. 59 shows (a) immunoblot analysis of the second cohort of tumors formed 5 weeks after implanting 786-0 cells expressing either H3-Cre or H4 Cre mixed 1:1 with RCC4 color switch reporter cells (b) Immunoblot analysis of tumors from the second cohort of 786-0 color switch reporter cells 5 weeks after implanting them on one flank and implanting 786-0 cells expressing either H3-Cre or H4 Cre on the opposite flank.
  • histone H3 histone H3
  • histone H3 histone H3
  • cell membranes to deliver proteins to which they are fused both in cell culture and in mice.
  • cell uptake does not require autophagy.
  • secreted histone H3 has specific posttranslational modifications that increase or decrease secretion and, without wishing to be bound by theory, transduction.
  • aspects of the invention can be used to systemically deliver large molecules, such as proteins, that have been designed to modulate intracellular targets or replace intracellular targets.
  • histone can refer to DNA binding structural proteins of chromosomes. Histones have a high proportion of positively charged amino acids such as lysine and arginine, which aids in DNA binding.
  • nucleosomal histones H2A, H2B, H3, H4 The five main types of histones fall into two groups: nucleosomal histones H2A, H2B, H3, H4; and HI histones.
  • the families H2A, H2B, H3, and H4 constitute the core histones, while the family of H1/H5 histones is known as the linker histones.
  • Each nucleosome core comprises 8 histones, two of each of the core histones, around which the DNA winds.
  • the linker histones thereby sit on top keeping the DNA in place and linking the nucleosomes to form higher-order structures.
  • the core histones are highly conserved across eukaryotes in terms of sequence and structure.
  • embodiments described herein can comprise histone polypeptides or fragments thereof from one or more eukaryotic species.
  • embodiments can comprise a histone polypeptide or fragment thereof corresponding to a mammalian histone.
  • Histone H2A can refer to to a variety of closely related proteins that vary' often by only a few amino acids. These can be further classified by the subfamilies Histone H2A F, 112 A 1 , and H2A 2 and include the specific histones H2AFBL H2AFB2, H2AFB3, H2AFJ, H2AFV, 11.2 AFX.
  • H2A consists of a main globular domain and a long N-terminal tail or C -terminal on one end of the molecule. Hie N-terminal tail or C -terminal tail is the common location of post- translational modification.
  • Histone H2FT also refers to a variety of closely related proteins further classified by the subfamilies H2BF, H2B1, and H2B2 and includes the specific exemplary histone proteins H2BFM, H2BFS, H2BFWT, H1ST1H2BA, HIST1H2BB, HIST1H2BC, HIST1H2BD, HIST1H2BE, HIST1H2BF, HIST1H2BG, HIST1H2BH, H1ST1H2B1, H1ST1H2BJ, BIST1 B2BK, F1IST1F12BL, BIST1 B2BM, HIST1H2BN, BIST1B2BO, and F1IST2F12BE.
  • Histone B3 refers to one of the fi ve main families of histone proteins and represents the most extensively modified of the histone proteins by post-translational modifications. Specific subfamilies of histone B3 include H3A1, H3A2, and B3A3 and include the exemplary B3 histones FIIST1FI3A, HIST1H3B, HIST1H3C, HIST1H3D,
  • FITST1FI3E FIIST1FI3F
  • HI5T1H3G ITIST1IT3FT
  • FIIST1FI3I FIIST1FI3I
  • FITST1FI3J TdIST2Td3C
  • HIST3H3 As the most modified of the histone proteins, 113 histones have emerged to play an important role in gene regulation and the emerging science of epigenetics.
  • the “Histone H4” family comprises the families H41 and H44 histones and includes the exemplary histones HXST1H4A, HIST1H4B, HTST1H4C, HTST1H4D, HIST1H4E, HIST1H4F, ! HS ! ! f !4G.
  • the histone can be a mammalian histone, polypeptide thereof, or fragment thereof.
  • polypeptide, ' “peptide” and “protein” can be used interchangeably herein and can refer to a polymer of amino acid residues. The terms can apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring ammo acid, as well as to naturally occurring ammo acid polymers and non-naturally occurring ammo acid polymer.
  • amino acid and amino acid residue can refer to natural amino acids, unnatural amino acids, and modified amino acids. Any reference to an amino acid, generally or specifically by name, includes reference to both the D and the L stereoisomers if their structure allows such stereoisomeric forms.
  • Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val).
  • Unnatural amino acids include, but are not limited to homolysine, homoarginine, homoserine, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4- aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3- aminoisbutyric acid, 2-aminopimelic acid, tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N- ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3 -hydroxy proline, 4- hydroxyproline, isodesmosine, allo-isoleucine, N-
  • Additional unnatural amino acids include modified amino acid residues which are chemically blocked, reversibly or irreversibly, or chemically modified on their N-terminal amino group or their side chain groups, as for example, N-methylated D and L amino acids or residues wherein the side chain functional groups are chemically modified to another functional group.
  • modified amino acids include methionine sulfoxide; methionine sulfone; aspartic acid-(beta- methyl ester), a modified amino acid of aspartic acid; N-ethylglycine, a modified amino acid of glycine; or alanine carboxamide, a modified amino acid of alanine Additional residues that can be incorporated are described in Sandberg et al., J. Med.
  • Amino acid analogs can refer to compounds that have the same basic chemical structure as a naturally occurring ammo acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an ammo group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfomum.
  • Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Ammo acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring ammo acid.
  • polypeptide can encompass a singular “polypeptide” as well as plural “polypeptides,” and can refer to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide can refer to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • polypeptides dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms.
  • polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
  • a polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specific nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
  • a polypeptide of the invention can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids.
  • Polypeptides can have a defined three- dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.
  • histone H3 includes the proteins of Swiss Prot AccNo. Q93081 (Histone H3), P68431 (Histone H3.1), Q71D13 (Histone H3.2), and P84243 (Histone H3.3) (each of which are incorporated by reference herein in their entireties with respect to the sequence itself) and variants thereof, including, but not limited to, the modified histone proteins thereof, as well as proteins which are substantially identical thereto, and, optionally, also lack the N-terminal methionine residue at position 1 of the sequences provided herein (e.g., a post-translational loss of the N-terminal methionine residue).
  • the histone polypeptide can comprise an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identical to Swiss Prot Acc No. Q93081 (Histone H3), P68431 (Histone H3.1), Q7D13 (Histone H3.2), or P84243 (Histone H3.3).
  • the histone polypeptide comprises an amino acid sequence according to (Start codon indicated by M - Met.):
  • nucleic acids or polypeptide sequences can refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a specified region). Percent identity can be measured, for example, using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters set, or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.”
  • the definition can also include sequences that have deletions and/or additions, as well as those that have substitutions. Algorithms can account for gaps and the like.
  • sequence comparison for example, one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are selected, if necessary, and sequence algorithm program parameters are selected. Default program parameters can be used, or alternative parameters can be selected.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • the histone polypeptide or fragment thereof can be a recombinant polypeptide.
  • the term “recombinant” can refer to genetic material (i.e., nucleic acids, the polypeptides they encode, and vectors and cells comprising such polynucleotides) that has been modified to alter its sequence or expression characteristics, such as by mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence to that of another gene, placing a gene under the control of a different promoter, expressing a gene in a heterologous organism, expressing a gene at a decreased or elevated levels, expressing a gene conditionally or constitutively in manner different from its natural expression profile, and the like.
  • Recombinant nucleic acids, polypeptides, and cells based thereon have been manipulated by man such that they are not identical to related nucleic acids, polypeptides, and cells found in nature.
  • the term “recombinant polypeptide” can refer to a polypeptide that is produced by recombinant techniques, wherein DNA or RNA encoding the expressed protein is inserted into a suitable expression vector that is in turn used to transform a host cell to produce the polypeptide.
  • Routine methods for making recombinant nucleic acids can be used to construct expression vectors encoding the polypeptides of interest using appropriate transcriptional/translational control signals and the protein coding sequences. (See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d Ed. (Cold Spring Harbor Laboratory 2001)).
  • the histone polypeptide or fragment thereof can be referred to as an “isolated polypeptide”.
  • isolated can refer to a molecule of interest (such as, for example, a polynucleotide or a polypeptide) that is in an environment different from that in which the molecule can naturally occur.
  • an “isolated” molecule is one which is substantially separated from the cellular components (e.g., membrane lipids, chromosomes, proteins) of the host cell from which it originated, or from the medium in which the host cell was cultured.
  • the term does not require that the biomolecule has been separated from all other chemicals or molecules, although certain isolated biomolecules can be purified to near homogeneity.
  • an “isolated polypeptide” can refer to a polypeptide that is free from at least one contaminating polypeptide or other contaminants that are found in its natural environment.
  • the isolated polypeptide is substantially free from any other contaminating polypeptides or other contaminants that are found in its natural environment which can interfere with its therapeutic, diagnostic, prophylactic or research use.
  • the histone polypeptide can comprise one or more modifications.
  • modification or “protein modification” can refer to one or more post-translational modifications or amino acid mutations (e.g., substitutions, deletions, and/or insertions), as is well understood in the art.
  • amino acid mutation can refer to one or more mutations of amino acid positions on a fragment of polypeptide and variants thereof, wherein the variant can be obtained by substituting, inserting or deleting amino acids at one or some sites on the polypeptide.
  • an amino acid mutation of the histone H3 polypeptide can comprise a substitution of Lys-14 with Gln-14 according to SEQ ID NO: 1, 2, or 3, such as to increase histone secretion and transduction. Substituting Lys-14 with Arg-14 prevents secretion and transduction, whereas changing Lys-14 to Gln-14 increases secretion and transduction.
  • amino acid mutations include a substitution of Lys-9 to Met-9 according to SEQ ID NO: 1, 2, or 3; Lys-14 to Gln-14 according to SEQ ID NO: 1, 2, or 3; Lys-27 to Met-27 according to SEQ ID NO: 1, 2, or 3; or any combination thereof.
  • the combination of amino acid mutations can comprise two or more substitutions, such as a substitution of Lys-14 to Gln-14, Lys-9 to Met-9, and Lys-27 to Met- 27.
  • substitution of Lys-14 to Gln-14 increases transfer.
  • substitution Lys-27 with Met-27 increases both secretion and transduction.
  • post-translational modifications can refer to one or more modifications that occur on a peptide after its translation by ribosomes is complete.
  • a post-translational modification can be a covalent modification and/or enzymatic modification.
  • the covalent modification can be catalyzed by an enzyme.
  • post-translation modifications include, but are not limited to, acylation, acetylation, alkylation (including methylation), biotinylation, butyrylation, carbamylation, carbonylation, deamidation, deiminiation, diphthamide formation, disulfide bridge formation, eliminylation, flavin attachment, formylation, gamma-carboxylation, glutamylation, glycylation, glycosylation, glypiation, heme C attachment, hydroxylation, hypusine formation, iodination, isoprenylation, lipidation, lipoylation, malonylation, methylation, myristolylation, oxidation, palmitoylation, pegylation, phosphopantetheinylation, phosphorylation, prenylation, propionylation,
  • a post-translational modification can include modifications of the amino terminus and/or the carboxyl terminus of a peptide.
  • Modifications of the terminal amino group include, but are not limited to, des-amino, N-lower alkyl, N-di- lower alkyl, and N-acyl modifications.
  • Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., wherein lower alkyl is C1-C4 alkyl).
  • a post- translational modification also includes modifications, such as but not limited to those described herein, of amino acids falling between the amino and carboxy termini.
  • the term post-translational modification can also include peptide modifications that include one or more detectable labels.
  • Modified histone proteins are well known in the art.
  • the suitable histone protein modifications on histone H3.1 can include, but are not limited to, any one or more listed herein.
  • the suitable histone protein modifications on a histone protein according to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a fragment or variant thereof can include, but are not limited to, any one or more listed herein.
  • Exemplary protein modifications for use according to the invention are set forth below. “Me” refers to methyl modification, “Ac” to acetyl modifications, “P” to phosphorylation modifications, and “Ub” to ubiquinylations. Modifications can be mono-, di-, or tri- modifications, such as mono-, di-, or tri-methylations.
  • the histone polypeptide can be acetylated at Lys-14 according to SEQ ID NO: 1, 2, or 3, or a fragment or variant thereof, such as to increase histone secretion and transduction.
  • Histone modifications can be reversibly controlled by enzymes.
  • histone acetylases are a class of enzymes that can attach an acetyl group to an amino acid on a histone protein
  • histone deacetylases are a class of enzymes that remove acetyl groups.
  • Embodiments described herein thus also comprise modified histone polypeptides and fragments thereof that comprise the loss of or reduced levels of one or more post-translational modification described herein. Referring to acetylation as the representative example, such a polypeptide can be referred to as hypoacetylated.
  • hypoacetylated such a polypeptide can be referred to as hypoacetylated.
  • the prefix “hypo-“ can refer to low levels of, or below, such as below normal.
  • Assays and methods to detect histone modifications are known in the art, including immunological methods and the like employing antibodies, apatamers, and immunologically active fragments of the antibodies that can bind to the histone modification of interest. Immunohistochemical and immunocytological methods can be used in detecting the modified histones or staining the cells to detect histone modifications, including the percent of cells staining for the modification. Mass spectroscopic and electrochemical means can also be used. Methods of detecting and measuring histone modifications including non-antibody- based protocols can be used, such as software programs providing for detection of epigenetic modifications.
  • Immunohistochemistry can refer to the use of antibodies or aptamers to detect proteins in biological samples such as cells and tissue sections. Methods described herein can be carried out, for example, using standard immunohistochemical techniques known in the art (reviewed in Gosling, Immunoassays: A Practical Approach, 2000, Oxford University Press). Detection can be accomplished by labeling a primary antibody or a secondary antibody with, for example, a radioactive isotope, a fluorescent label, an enzyme or any other detectable label known in the art. Visual grading of tissue sections by intensity of staining is well known in the art. Standard controls from tumor and healthy tissue samples can be used to control for variation among samples and reagents. Moreover, negative controls that do not include primary antibodies specific for the target (i.e., histone) can be used as controls.
  • the polypeptide can comprise a fragment polypeptide (i.e., “fragment”, “variant”, or “derivative”), which can refer to a short amino acid sequence of a larger polypeptide.
  • Protein fragments can be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part of.
  • Representative examples of polypeptide fragments of the invention include, for example, fragments comprising about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, about 100, about 200, and about 500 amino acids or more in length.
  • polypeptides and fragments thereof retain at least some biological activity.
  • Polypeptides as described herein can include fragment, variant, or derivative molecules without limitation, so long as the polypeptide still serves its function.
  • polypeptide fragments can include proteolytic fragments, deletion fragments and fragments which more easily reach the site of action when delivered to an animal.
  • Polypeptides and fragments thereof can comprise variant regions, including fragments as described herein, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can occur naturally, such as an allelic variant.
  • allelic variant is intended alternate forms of a gene occupying a given locus on a chromosome of an organism.
  • Non-naturally occurring variants can be produced using art-known mutagenesis techniques.
  • Polypeptides and fragments thereof can comprise conservative or nonconservative amino acid substitutions, deletions or additions.
  • Polypeptides or fragments thereof can also include derivative molecules.
  • a “derivative” of a polypeptide or a polypeptide fragment can refer to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group.
  • derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.
  • the fragment can be an N-terminal fragment of histone H3, a C- terminal fragment of histone H3, or an internal fragment of histone H3 (wherein a portion of the N-terminus and a portion of the C-terminus are truncated, thus leaving a fragment corresponding to a polypeptide sequence that is internal to the full length histone H3 polypeptide).
  • deletion of the N-terminal tail of histone H3 increases levels of extracellular H3 (i.e., histone H3 secreted from the cell).
  • the fragment of histon H3 comprises a deletion of about 5 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, about 10 amino acids from SEQ
  • the fragment polypeptide comprises a fragment of an amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identical thereto.
  • the fragment can be about 5 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 5 amino acids to about 10 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 10 amino acids to about 20 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 20 amino acids to about 30 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 30 amino acids to about 40 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 40 amino acids to about 50 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 50 amino acids to about 60 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 60 amino acids to about 70 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 70 amino acids of SEQ
  • the histone polypeptide or fragment thereof exhibits at least one functional and/or biological activity.
  • biological activity can refer to an in vivo activity, such as an activity within an organism or within a cell. Examples of such biological activities include, but are not limited to, crossing a cell membrane, secretion from a cell, uptake by a cell, or delivery of a macromolecule.
  • the histone polypeptide or fragment thereof can be isolated from a host cell, such as a host cell which comprises a nucleic acid encoding for and expressing the polypeptide or fragment thereof.
  • a host cell such as a host cell which comprises a nucleic acid encoding for and expressing the polypeptide or fragment thereof.
  • the phrase "host cell”, “host cell line,” and “host cell culture” can be used interchangeably and can refer to the cells into which exogenous nucleic acid can be introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and the progeny derived therefrom without regard to the number of passages. The progeny does not need to be completely identical in nucleic acid content to a parent cell, but it can contain mutations.
  • Host cells can include any eukaryotic cell or prokaryotic cell, including but not limited to a mammalian cell, avian cell, amphibian cell, plant cell, fish cell, insect cell, bacterial cell, yeast cell, whether in vitro or in vivo.
  • the host cell can be a mammalian cell.
  • mammalian cell can refer to a cell of any mammal, including humans.
  • the phrase can refer to cells in vivo, such as, for example, in an organism or in an organ of an organism.
  • the phrase also can refer to cells in vitro, such as, for example, cells maintained in cell culture.
  • the histone polypeptide or fragment thereof can be component of a fusion protein.
  • fusion protein” or “fusion polypeptide” can refer to a complex formed by the fusion of a polypeptide sequence (i.e., a histone polypeptide sequence) with one or more elements.
  • the fusion protein can comprise the histone polypeptide or fragment thereof fused to at least one functional moiety.
  • the phrase “functional moiety” can refer to an element (i.e., component) that can perform a function, for example, an imaging function, a biological function (e.g., replacing a biological function that is otherwise missing or erroneously modulated, such as due to a missing or defective protein), or a drug- delivery function.
  • functional moieties comprise a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof .
  • therapeutic moiety can refer to molecule, compound, or fragment thereof that is used for the treatment of a disease or for improving the well-being of an organism or that otherwise exhibit healing power (e.g., pharmaceuticals, drugs, and the like).
  • a therapeutic moiety can be a chemical, or fragment thereof, of natural or synthetic origin used for its specific action against disease, for example cancer.
  • Non- limiting examples of therapeutic moieties include a therapeutic protein or polypeptide, a small molecule, or a toxin.
  • therapeutic moieties include chemotherapeutic agents, such as those described herein.
  • therapeutic moieties can replace a biological function that is otherwise missing or erroneously modulated, such as due to a missing or defective protein.
  • a therapeutic moiety can replace a missing or defective tumor suppressor protein in cancer (e.g., p53 or pVHL), or replace a missing or defective enzyme in metabolic diseases.
  • cytotoxic moiety can refer to molecule, compound, or fragment thereof that has a toxic or poisonous effect on cells, or that kills cells.
  • Chemotherapy and radiotherapy are forms of cytotoxic therapy. Treating cells with a cytotoxic moiety can produce a variety of results cells can undergo necrosis, stop actively growing and dividing, or activate a genetic program of controlled cell death (i.e., apoptosis).
  • cytotoxic moieties include, but are not limited to, SN-38, bendamustine, VDA, doxorubicin, pemetrexed, vorinostat, lenalidomide, irinotecan, ganetespib, docetaxel, 17-AAG, 5-FU, abiraterone, crizotinib, KW-2189, BUMB2, DC1, CC-1065, adozelesin or fragments ) thereof.
  • imaging moiety can refer to a molecule, compound, or fragment thereof that facilitates a technique and/or process used to create images or take measurements of a cell, tissue, and/or organism (or parts or functions thereof) for clinical and/or research purposes.
  • An imaging moiety can produce, for example, a signal through emission and/or interaction with electromagnetic, nuclear, and/or mechanical (e.g., acoustic as in ultrasound) energy.
  • An imaging moiety can be used, for example, in various radiology, nuclear medicine, endoscopy, thermography, photography, spectroscopy, and microscopy methods.
  • the imaging moiety i.e., the diagnostic agent, or imaging agent
  • a nonlimiting example of an imaging moiety comprises one which can be used for PET imaging, such as HSV TK, or fluorescence imaging, such as GFP.
  • the imaging moiety can comprise a “label” or “detectable moiety”.
  • a “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (for example, those as used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
  • Labels can be conjugated directly to a biorecognition molecules, or to probes that bind these molecules, using conventional methods that are well known in the arts. Multiple labeling schemes are known in the art and permit a plurality of binding assays to be performed simultaneously. Different labels can be radioactive, enzymatic, chemiluminescent, fluorescent, quantum dot, or others. Methods of covalently or noncovalently conjugating labels to antibodies are well known to one of ordinary skill in the art. Methods of detecting proteins and modified proteins by use of labeled antibodies are also well known to persons of ordinary skill in the art.
  • linker can refer to a chemical moiety or bond that attaches two or more molecules.
  • the two or more molecules can comprise a drug delivery moiety (i.e., a histone polypeptide or fragment thereof), a functional moiety, and/or a solid support (e.g., microparticle).
  • Linkers can comprise contiguous chains of one or more components, such as carbon, oxygen, nitrogen, sulfur, phosphorous and combinations thereof.
  • Linkers can connect the molecule via a covalent bond or other means, such as ionic or hydrogen bond interactions.
  • Linkers can be cleavable, such as to release a functional moiety from the histone polypeptide once inside a target cell.
  • the linker can comprise the nucleic acid sequence:
  • linker spacer group can refer to atoms in the linker that provide space between the two molecules joined by the linker.
  • the linker comprises from 1 to 30 or less amino acids linked by peptide bonds.
  • the amino acids can be selected from the 20 naturally occurring amino acids. Alternatively, non-natural amino acids can be incorporated by chemical synthesis, post- translational chemical modification or by in vivo incorporation by recombinant expression in a host cell. Some of these amino acids can be glycosylated.
  • amino acids of the linker are selected from glycine, alanine, proline, asparagine, glutamine, lysine, aspartate, and glutamate.
  • linker can be made up of a majority of amino acids that are sterically unhindered, such as glycine, alanine and/or serine.
  • linkers can comprise polyglycine linkers, including but not limited to (Gly)3, (GlyMSEQ ID NO:5), (Gly)s (SEQ ID NO: 6).
  • Other examples include polyalanines, poly(Gly-Ala), poly(GlymSer), poly (Glvn-Glu), poly(Glyn-Lys), poly(Gly n - Asp), and poly(Gly n -Arg) motifs.
  • Other specific examples of linkers are (GlyjsLysiGiy ) * (SEQ ID NO; 7); (GlyjsAsnGlySerlGlyjz (SEQ ID NO: 8);
  • the peptide linker is selected from the group consisting of a glycine rich peptide, e.g.
  • Gly-Gly- Gly the sequences [Gly-Ser]r.(SEQ ID NO: 11), [Gly-Gly-Serjn (SEQ ID NO: 12), [Gly-Gly- Gly-Ser]n (SEQ ID NO: 13) and [Gly-Gly-Gly-Giy-Ser]n (SEQ ID NO: 14), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, [Gly-Giy-Gly Ser] i (SEQ ID NO: 15), [Gly-Gly-Gly-Gly Ser] 1 (SEQ ID NO: 16), [Gly-Gly-Gly Serb (SEQ ID NO: 17), or [Gly-Gly-Gly-Gly-Gly Ser] 3 (SEQ ID NO: 18).
  • charged linkers can be used.
  • Such charges linkers can contain a significant number of acidic residues (e.g.. Asp, Glu, and the like), or can contain a significant number of basis residues (e.g., Lys, Arg, and the like), such that the linker has a pi lower than 7 or greater than 7, respectively.
  • acidic residues e.g.. Asp, Glu, and the like
  • basis residues e.g., Lys, Arg, and the like
  • Such linkers can impart advantages to the engineered polypeptides disclosed herein, such as improving solubility and/or stability characteristics of such polypeptides at a specific pH, such as a physiological pH (e.g., between pH 7.2 and pH 7.6, inclusive), or a pH of a pharmaceutical composition comprising such polypeptides.
  • a specific pH such as a physiological pH (e.g., between pH 7.2 and pH 7.6, inclusive), or a pH of a pharmaceutical composition comprising such polypeptides.
  • an "acidic linker’ ' is a linker that has a pi of less than 7; between 6 and 7, inclusive; between 5 and 6, inclusive; between 4 and 5, inclusive, between 3 and 4, inclusive; between 2 and 3, inclusive, or between I and 2, inclusive.
  • a “ basic linker’ is a linker that has a pi of greater than 7, between 7 and 8, inclusive; between 8 and 9, inclusive; between 9 and 10, inclusive; between 10 and i t, inclusive; between 11 and 12 inclusive, or between 12 and 13, inclusive.
  • an acidic linker will contain a sequence that is selected from the group consisting of [Gly ⁇ Glu]a(SEQ ID NO; 19); [Gly ⁇ Gly ⁇ Glu]n (SEQ ID NO; 20): [Gly-Gly-Gly-GIu],, (SEQ ID MO; 21); [Gly-GIy-Gly-Gly- Giujn (SEQ ID NO; 22), [Gly ⁇ Asp]GSEQ ID NO: 23); [Gly-GIy-Asp] n (SEQ ID NO: 24); [Gly-Gly-Gly-Asp]n(SEQ ID NO: 24); [Giy-Gly-Gly-Gly-Asp]n(SEQ ID NO: 26) where n is I, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; for example, [Gly-Gly-Gluje (SEQ ID NO: 27).
  • a basic linker will contain a sequence that is selected from the group consisting of [Gly-Lys];; (SEQ ID NO: 28); [Gly-Gly-Lys] «(SEQ ID NO: 29); [Giy-Gly-Gly-Lys] n (SEQ ID NO: 30); [Gly-Gly-Gly-Gly-Lysjr, (SEQ ID NO: 31), [Gly-Arg] « (SEQ ID NO: 32); [Gly- Giy-Arg] :; (SEQ ID NO: 33); [Gly-Gly-Gly-Arg] K (SEQ ID NO: 34); [Gly-Gly-Gly-Giy- Arg] n (SEQ ID NO: 35) where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; for example, [Gly-Gly- Lysjs (SEQ ID NO: 36).
  • linkers can be prepared which possess certain structural motifs or characteristics, such as an a helix.
  • a linker can contain an sequence that is selected from the group consisting of [Glu-A3a-Ala-Aia-Lys]n(SEQ ID NO: 37), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, for example, [Giu- A1 a-Ala- A! a-Ly s] 3 (SEQ ID NO: 38), [GIu-A3a ⁇ Ala-A3a ⁇ Lys]i (SEQ ID NO; 39), or [G3 u- A3 a- Ala- Ai a-Ly s ] 5 (SEQ ID NO: 40).
  • a non-peptidic linker can be employed to serve as the LI moiety of an engineered polypeptide described herein.
  • an exemplary non-peptide linker such as a PEG linker can be so-employed. See, e g., W02000024782.
  • a PEG linker has a molecular weight ofl kDa to 1000 kDa.
  • linkers suitable for use in accordance with the invention can possess one or more of the characteristics and motif, such as those described herein.
  • a linker can comprise an acidic linker as well as a structural motif, such as an alpha helix.
  • a linker can comprise a basic linker and a structural motifi such as an alpha helix.
  • a linker can comprise an acidic linker, a basic linker, and a structural motif, such as an a helix.
  • engineered polypeptides in accordance with the invention can possess more than one linker, and each such linker can possess one or more of the characteristics described herein.
  • linkers described herein are exemplary', and linkers within the scope of this invention can be much longer and can include other residues.
  • immune adjuvant can refer to any substance that assists or modifies the action of a pharmaceutical, including but not limited to immune adjuvants that enhance and/or diversify the immune response to an antigen.
  • immune adjuvants include compounds that can enhance the immune response to an antigen.
  • An immune adjuvant can enhance body fluid and/or cellular immunity. Substances that stimulate the innate immune response are included within the definition of immune adjuvant herein.
  • the functional moiety can be a macromolecule.
  • the term “macromolecule” can refer to peptides, proteins, and large molecules (molecular weights of 1000 Daltons or more).
  • macromolecules include proteins (e.g., polypeptides), fats, fatty acids or nucleic acids (e.g., oligonucleotides and polynucleotides), of biological or synthetic origin.
  • engineered polypeptides described herein have delivered cargo such as Cre recombinase (38kDa), eGFP (26.9kDa), and mCherry (28kDa). See, for example, the sequences according to SEQ ID NO: 43-SEQ ID NO: 48.
  • the macromolecule can be a protein or a fragment thereof.
  • the protein can be an antibody or fragment thereof, transcription factor or other protein with biological activity, a growth inhibitor (i.e., suppresses in vitro or in vivo cell growth) or a cytokine.
  • the term “antibody” can refer to a polypeptide from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the antibody or fragment thereof can be specific for an oncoprotein, such as c-Myc.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains can be classified as kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes,
  • the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the teens variable light chain (VL) and variable heavy chain (VH) can refer to these light and heavy chains respectively.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments, such as produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond.
  • the F(ab)'2 can be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments can be synthesized de novo, such as chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments, such as those produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)). For example, a single chain antibody can be fused to a histone polypeptide, such as histone H3.
  • a histone polypeptide such as histone H3.
  • cytokine can refer to a molecule that mediates and/or regulates a biological or cellular function or process (e.g. immunity, inflammation, and hematopoiesis).
  • cytokine includes “lymphokines,” “chemokines,” “monokines,” and “interleukins”.
  • cytokines examples include, but are not limited to, GM-CSF, IL-la, IL-10, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IFN-a, IFN-b, IFN-g, MIP-la, MIR-Ib, TGF-b, TNF-a, and TNF-b.
  • cytokine as used herein is meant to also include cytokine variants comprising one or more amino acid mutations in the amino acid sequences of the corresponding wild-type cytokine, such as for example the IL-2 variants described in Sauve et al., Proc Natl Acad Sci USA 88, 4636-40 (1991); Hu et al., Blood 101, 4853-4861 (2003) and US Pat. Publ. No. 2003/0124678; Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000); Heaton et al., Cancer Res 53, 2597-602 (1993) and U.S. Pat. No. 5,229,109; US Pat. Publ. No. 2007/0036752; WO 2008/0034473; WO 2009/061853; or in WO 2012/107417.
  • nucleic acids encoding a histone polypeptide or fragment thereof.
  • the phrase “nucleic acid” can refer to deoxyribonucleotides or ribonucleotides and polymers thereof, such as those in single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base, such as a purine base or pyrimidine base.
  • the term also encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • a nucleic acid sequence can also encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the reference sequence explicitly indicated.
  • the human histone H3 nucleic acid sequence comprises
  • oligonucleotide can refer to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, for example more than three, and, in embodiments, more than ten. There is no precise upper limit on the size of an oligonucleotide. In embodiments, an oligonucleotide can be shorter than about 250 nucleotides, shorter than about 200 nucleotides, or shorter than about 100 nucleotides. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide.
  • the oligonucleotide can be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.
  • the nucleic acid comprises a nucleotide sequence according to 4 or a fragment thereof, or a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identical thereto.
  • the nucleic acid comprises a “nucleic acid fragment”, which can refer to a portion of a larger nucleic acid molecule.
  • a cell comprising the nucleic acids described herein.
  • the cell can be referred to as a host cell.
  • the phrase "host cell”, “host cell line,” and “host cell culture” can be used interchangeably and can refer to the cells into which exogenous nucleic acid can be introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and the progeny derived therefrom without regard to the number of passages. The progeny does not need to be completely identical in nucleic acid content to a parent cell, but it can contain mutations.
  • Host cells can include any eukaryotic cell or prokaryotic cell, including but not limited to a mammalian cell, avian cell, amphibian cell, plant cell, fish cell, insect cell, bacterial cell, yeast cell, whether in vitro or in vivo.
  • the host cell can be a mammalian cell.
  • mammalian cell can refer to a cell of any mammal, including humans.
  • the phrase can refer to cells in vivo, such as, for example, in an organism or in an organ of an organism.
  • the phrase also can refer to cells in vitro, such as, for example, cells maintained in cell culture.
  • the engineered polypeptides described herein can be designed at the amino acid level. These sequences can then be back translated using a variety of software products known in the art such that the nucleotide sequence is optimized for expression, such as in a given host (for example, based protein expression, codon optimization, restriction site content). For example, the nucleotide sequence can be optimized for E. coli based protein expression and for restriction site content. Based on the nucleotide sequence of interest, overlapping oligonucleotides can be provided for multistep PCR, as known in the art. These oligonucleotides can be used in multiple PCR reactions under conditions well known in the art to build the cDNA encoding the protein of interest.
  • For one example is 1 x Amplitaq Buffer, 1.3 mM MgC12, 200 uM dNTPs, 4 U Amplitaq Gold, 0.2 uM of each primer (AmpliTaq Gold, ABI), with cycling parameters: (94 C:30 s, 58 C:1 min, 72 C: 1 min), 35 cycles.
  • cycling parameters (94 C:30 s, 58 C:1 min, 72 C: 1 min), 35 cycles.
  • Restriction sites can be added to the ends of the PCR products for use in vector ligation as known in the art.
  • Specific sites can include Ndel and Xhol, such that the cDNA can then be in the proper reading frame in a pET45b expression vector (Novagen). By using these sites, any N-terminal His Tag that are in this vector can be removed as the translation start site can then be downstream of the tag.
  • verification can be conduct by sequencing using e.g., T7 promoter primer, T7 terminator primer and standard ABI BigDye Term v3.1 protocols as known in the art.
  • Sequence information can be obtained from e.g., an ABI 3730 DNA Analyzer and can be analyzed using Vector NTI v.10 software (Invitrogen).
  • Expression constructs can be designed in a modular manner such that linker sequences can be easily cut out and changed, as known in the art. The skilled artisan will recognize that expression constructs can also be designed without the use of restriction enzymes, such as using homology based methods like In-Fusion Cloning and Gibson Assembly.
  • Protease recognition sites can be incorporated into constructs useful for the design, construction, manipulation and production of recombinant engineering polypeptides described herein.
  • the engineered polypeptides described herein can be prepared using biological, chemical, and/or recombinant DNA techniques that are known in the art. Exemplary methods are described herein and in U.S. Pat. No. 6,872,700; WO 2007/139941; WO 2007/140284; WO 2008/082274; WO 2009/011544; and US Publication No. 2007/0238669, the disclosures of which are incorporated herein by reference in their entireties. Other methods for preparing the compounds are set forth herein.
  • the engineered polypeptides described herein can be prepared using standard solid-phase peptide synthesis techniques, such as an automated or semiautomated peptide synthesizer. For example, using such techniques, an alpha-N-carbamoyl protected amino acid and an amino acid attached to the growing peptide chain on a resin are coupled at RT in an inert solvent (e.g., dimethylformamide, N-methylpyrrolidinone, methylene chloride, and the like) in the presence of coupling agents (e.g., dicyclohexylcarbodiimide, 1-hydroxybenzo- triazole, and the like) in the presence of a base (e.g., diisopropylethylamine, and the like).
  • an inert solvent e.g., dimethylformamide, N-methylpyrrolidinone, methylene chloride, and the like
  • coupling agents e.g., dicyclohexylcarbodiimide, 1-hydroxybenzo- tri
  • the alpha-N-carbamoyl protecting group is removed from the resulting peptide-resin using a reagent (e.g., trifluoroacetic acid, piperidine, and the like) and the coupling reaction repeated with the next N-protected amino acid to be added to the peptide chain.
  • a reagent e.g., trifluoroacetic acid, piperidine, and the like
  • Suitable N-protecting groups are well known in the art, such as t-butyloxy carbonyl (tBoc) fluorenylmethoxycarbonyl (Fmoc), and the like.
  • tBoc t-butyloxy carbonyl
  • Fmoc fluorenylmethoxycarbonyl
  • the solvents, amino acid derivatives and 4- methylbenzhydryl-amine resin used in the peptide synthesizer can be purchased from Applied Biosystems Inc. (Foster City, Calif.).
  • Solid phase peptide synthesis can be used for the engineered polypeptides, since in general solid phase synthesis is a straightforward approach with excellent scalability to commercial scale, and can be compatible with relatively long engineered polypeptides.
  • Solid phase peptide synthesis can be carried out with an automatic peptide synthesizer (Model 430A, Applied Biosystems Inc., Foster City, Calif.) using the NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry (See Applied Biosystems User's Manual for the ABI 430A Peptide Synthesizer, Version 1.3B Jul. 1, 1988, section 6, pp.
  • Boc-peptide-resins can be cleaved with HF (-5° C. to 0° C., 1 hour).
  • the peptide can be extracted from the resin with alternating water and acetic acid, and the filtrates lyophilized.
  • the Fmoc-peptide resins can be cleaved according to standard methods (e.g., Introduction to Cleavage Techniques,
  • Peptides can also be assembled using an Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.).
  • Non-peptide compounds can be prepared by art-known methods.
  • phosphate-containing amino acids and peptides containing such amino acids can be prepared using methods known in the art, such as described in Bartlett et al, 1986, Biorg. Chem. 14:356-377.
  • the engineered polypeptides can alternatively be produced by recombinant techniques well known in the art. See, e.g., Sambrook et ak, 1989 (Id.). These engineered polypeptides produced by recombinant technologies can be expressed from a polynucleotide.
  • polynucleotides including DNA and RNA, that encode such engineered polypeptides can be obtained from the wild-type cDNA, taking into consideration the degeneracy of codon usage, and can be further engineered to incorporate the indicated substitutions.
  • These polynucleotide sequences can incorporate codons facilitating transcription and translation of mRNA in microbial hosts.
  • Non-peptide compounds useful in the invention can be prepared by art-known methods. For example, phosphate-containing amino acids and peptides containing such amino acids can be prepared using methods known in the art. See, e.g., Bartlett and Landen, 1986, Bioorg. Chem. 14: 356- 77.
  • a variety of expression vector/host systems can be utilized to contain and express an engineered polypeptide coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,
  • Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), WI 38, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols for the recombinant expression of the protein are described herein and/or are known in the art.
  • polynucleotide sequences are useful in generating new and useful viral and plasmid DNA vectors, new and useful transformed and transfected prokaryotic and eukaryotic host cells (including bacterial, yeast, and mammalian cells grown in culture), and new and useful methods for cultured growth of such host cells that can express the engineered polypeptides.
  • the polynucleotide sequences encoding engineered polypeptides herein can be useful for gene therapy in instances where underproduction of engineered polypeptides can be alleviated, or the need for increased levels of such can be met.
  • This invention also provides for processes for recombinant DNA production of the engineered polypeptides.
  • a process for producing the engineered polypeptides from a host cell containing nucleic acids encoding the engineered polypeptide comprising: (a) culturing the host cell containing polynucleotides encoding the engineered polypeptide under conditions facilitating the expression of the DNA molecule; and (b) obtaining the engineered polypeptide.
  • Host cells can be prokaryotic or eukaryotic and include bacteria, mammalian cells (such as Chinese Hamster Ovary (CHO) cells, monkey cells, baby hamster kidney cells, cancer cells or other cells), yeast cells, and insect cells.
  • mammalian cells such as Chinese Hamster Ovary (CHO) cells, monkey cells, baby hamster kidney cells, cancer cells or other cells
  • yeast cells such as yeast cells, and insect cells.
  • Mammalian host systems for the expression of the recombinant protein also are well known to those of skill in the art. Host cell strains can be chosen for their ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cleaves a “prepro” form of the protein, can also be important for correct insertion, folding and/or function. Different host cells, such as CHO, HeLa, MDCK, 293, W138, and the like, have specific cellular machinery and characteristic mechanisms for such post-translational activities, and can be chosen to ensure the correct modification and processing of the introduced foreign protein.
  • a yeast system can be employed to generate the engineered polypeptides of the invention.
  • the coding region of the engineered polypeptides DNA is amplified by PCR.
  • a DNA encoding the yeast pre-pro-alpha leader sequence is amplified from yeast genomic DNA in a PCR reaction using one primer containing nucleotides 1-20 of the alpha mating factor gene and another primer complementary to nucleotides 255-235 of this gene (Kurjan and Herskowitz, 1982, Cell, 30:933-43).
  • the pre-pro-alpha leader coding sequence and engineered polypeptide coding sequence fragments are ligated into a plasmid containing the yeast alcohol dehydrogenase (ADH2) promoter, such that the promoter directs expression of a fusion protein consisting of the pre-pro-alpha factor fused to the mature engineered polypeptide.
  • ADH2 yeast alcohol dehydrogenase
  • the vector further includes an ADH2 transcription terminator downstream of the cloning site, the yeast “2-micron” replication origin, the yeast leu-2d gene, the yeast REP1 and REP2 genes, the E.
  • the coli beta-lactamase gene and an E. coli origin of replication.
  • the beta-lactamase and leu-2d genes provide for selection in bacteria and yeast, respectively.
  • the leu-2d gene also facilitates increased copy number of the plasmid in yeast to induce higher levels of expression.
  • the REPl and REP2 genes encode proteins involved in regulation of the plasmid copy number.
  • the DNA construct described in the preceding paragraph is transformed into yeast cells using a known method, e.g., lithium acetate treatment (Steams et al., 1990, Meth. Enz. 185: 280-297).
  • the ADH2 promoter is induced upon exhaustion of glucose in the growth media (Price et al., 1987, Gene 55:287).
  • the pre-pro-alpha sequence effects secretion of the fusion protein from the cells.
  • the yeast KEX2 protein cleaves the pre-pro sequence from the mature engineered polypeptides (Bitter et al., 1984, Proc. Natl. Acad. Sci. USA 81:5330-5334).
  • Engineered polypeptides of the invention can also be recombinantly expressed in yeast, e.g., Pichia, using a commercially available expression system, e.g., the Pichia Expression System (Invitrogen, San Diego, Calif.), following the manufacturer's instructions. This system also relies on the pre-pro-alpha sequence to direct secretion, but transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction by methanol.
  • AOX1 alcohol oxidase
  • the secreted engineered polypeptide is purified from the yeast growth medium by, e.g., the methods used to purify said engineered polypeptide from bacterial and mammalian cell supernatants.
  • the DNA encoding an engineered polypeptide can be cloned into a baculovirus expression vector, e.g. pVL1393 (PharMingen, San Diego, Calif.).
  • This engineered-polypeptide-encoding vector is then used according to the manufacturer's directions (PharMingen) or known techniques to infect Spodoptera frugiperda cells, grown for example in sF9 protein-free media, and to produce recombinant protein.
  • the protein is purified and concentrated from the media using methods known in the art, e.g.
  • heparin- Sepharose column Pharmacia, Piscataway, N.J.
  • sequential molecular sizing columns Amicon, Beverly, Mass.
  • resuspended in appropriate solution e.g. PBS.
  • SDS-PAGE analysis can be used to characterize the protein, for example by showing a single band that confirms the size of the engineered polypeptide, as can full amino acid amino acid sequence analysis, e.g. Edman sequencing on a Proton 2090 Peptide Sequencer, or confirmation of its N-terminal sequence.
  • the DNA sequence encoding the predicted mature engineered polypeptide can be cloned into a plasmid containing a promoter and, optionally, a leader sequence (see, e.g., Better et ak, 1988, Science 240:1041-1043). The sequence of this construct can be confirmed by automated sequencing.
  • the plasmid can then be transformed into E. coli, strain MCI 061, using standard procedures employing CaC12 incubation and heat shock treatment of the bacteria (Sambrook et ak, Id.). The transformed bacteria are grown in LB medium supplemented with carbenicillin, and production of the expressed protein is induced by growth in a suitable medium.
  • the leader sequence can affect secretion of the mature engineered polypeptide and be cleaved during secretion.
  • the secreted recombinant engineered polypeptide can be purified from the bacterial culture media by the method described herein.
  • the engineered polypeptides can be expressed in an insect system.
  • Insect systems for protein expression are well known to those of skill in the art.
  • Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the engineered polypeptide coding sequence is cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • Successful insertion of an engineered polypeptide will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat.
  • the recombinant viruses are then used to infect S.
  • frugiperda cells or Trichoplusia larvae in which engineered polypeptide of the invention is expressed Smith et al., 1983, J. Virol. 46:584; Engelhard et al., 1994, Proc. Natl. Acad. Sci. USA 91:3224-3227).
  • the DNA sequence encoding the engineered polypeptides can be amplified by PCR and cloned into an appropriate vector, for example, pGEX-3X (Pharmacia, Piscataway, N. J.).
  • the pGEX vector is designed to produce a fusion protein comprising glutathione-S-transferase (GST), encoded by the vector, and a protein encoded by a DNA fragment inserted into the vector's cloning site.
  • the primers for the PCR can be generated to include, for example, an appropriate cleavage site.
  • the recombinant fusion protein can then be cleaved from the GST portion of the fusion protein.
  • the pGEX- 3X/engineered polypeptide construct is transformed into E. coli XL-1 Blue cells (Stratagene, La Jolla, Calif.), and individual transformants are isolated and grown at 37° C. in LB medium (supplemented with carbenicillin) to an optical density at wavelength 600 nm of 0.4, followed by further incubation for 4 hours in the presence of 0.5 mM Isopropyl beta-D- thiogalactopyranoside (Sigma Chemical Co., St. Louis, Mo.). Plasmid DNA from individual transformants is purified and partially sequenced using an automated sequencer to confirm the presence of the engineered polypeptide-encoding gene insert in the proper orientation.
  • the fusion protein when produced as an insoluble inclusion body in the bacteria, can be purified as described herein or as follows. Cells are harvested by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/mL lysozyme (Sigma Chemical Co.) for 15 min. at RT. The lysate is cleared by sonication, and cell debris is pelleted by centrifugation for 10 min. at 12,000xg. The fusion protein- containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, layered over 50% glycerol, and centrifuged for 30 min.
  • the pellet is resuspended in standard phosphate buffered saline solution (PBS) free of Mg++ and Ca++.
  • PBS phosphate buffered saline solution
  • the fusion protein is further purified by fractionating the resuspended pellet in a denaturing SDS polyacrylamide gel (Sambrook et al., supra). The gel is soaked in 0.4 M KC1 to visualize the protein, which is excised and electroeluted in gel-running buffer lacking SDS. If the GST/engineered polypeptide fusion protein is produced in bacteria as a soluble protein, it can be purified using the GST Purification Module (Pharmacia Biotech).
  • the fusion protein can be subjected to digestion to cleave the GST from the mature engineered polypeptide.
  • the digestion reaction (20-40 pg fusion protein, 20-30 units human thrombin (4000 U/mg (Sigma) in 0.5 mL PBS) is incubated 16-48 hrs. at RT and loaded on a denaturing SDS-PAGE gel to fractionate the reaction products. The gel is soaked in 0.4 M KC1 to visualize the protein bands.
  • the identity of the protein band corresponding to the molecular weight of the engineered polypeptide can be confirmed by partial amino acid sequence analysis using an automated sequencer (Applied Biosystems Model 473A, Foster City, Calif.).
  • 293 cells can be co-transfected with plasmids containing the engineered polypeptides cDNA in the pCMV vector (5' CMV promoter, 3' HGH poly A sequence) and pSV2neo (containing the neo resistance gene) by the calcium phosphate method.
  • the vectors can be linearized with Seal prior to transfection.
  • an alternative construct using a similar pCMV vector with the neo gene incorporated can be used.
  • Stable cell lines are selected from single cell clones by limiting dilution in growth media containing 0.5 mg/mL G418 (neomycin-like antibiotic) for 10-14 days. Cell lines are screened for engineered polypeptides expression by ELISA or Western blot, and high- expressing cell lines are expanded for large scale growth.
  • G418 neomycin-like antibiotic
  • viral delivery systems can be used to transduce cells.
  • lentiviral infection i.e., lentiviral transduction
  • lentiviral transduction can be used to introduce an exogenous polynucleotide into the genome of a cell.
  • the use of lentiviral vectors permits stable expression of the polynucleotide of interest.
  • lentiviral transduction can be performed by incubating cells to be transduced with a lentiviral vector carrying at least one polynucleotide encoding a polypeptide of interest.
  • the transformed cells are used for long-term, high-yield protein production and as such stable expression is desirable.
  • the cells can be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the selectable marker can be designed to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell.
  • a number of selection systems can be used to recover the cells that have been transformed for recombinant protein production.
  • selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside, G418; also, that confers resistance to chlorsulfuron; hygro, that confers resistance to hygromycin; or PAC, that confers resistance to puromycin.
  • Additional selectable genes that can be useful include trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine.
  • Markers that give a visual indication for identification of transformants include anthocyanins, beta- glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin.
  • the engineered polypeptides of the invention can be produced using a combination of both automated peptide synthesis and recombinant techniques.
  • Engineered polypeptides can be made synthetically or recombinantly and then, in embodiments, ligated together using methods known in the art, such as “native chemical ligation” and known variations thereof in which an amide bond is formed joining the parent compounds. See for example U.S. Pat. No. 6,326,468.
  • an engineered polypeptide of the invention can contain a combination of modifications including deletion, substitution, insertion and derivatization by PEGylation (or other moiety, e.g. polymer, fatty acyl chain, C-terminal amidation).
  • Such an engineered polypeptide can be produced in stages.
  • an intermediate engineered polypeptide containing the modifications of deletion, substitution, insertion, and any combination thereof can be produced by recombinant techniques as described.
  • the intermediate engineered polypeptide is PEGylated (or subjected to other chemical derivatization, e.g., acylation, C-terminal amidation) through chemical modification with an appropriate PEGylating reagent (e.g., fromNeKtar Transforming Therapeutics, San Carlos, Calif.) to yield the engineered polypeptide derivative.
  • an appropriate PEGylating reagent e.g., fromNeKtar Transforming Therapeutics, San Carlos, Calif.
  • C-terminal amidation can be achieved by use of a glycine amino acid-C-terminally extended precursor, synthesized for example in yeast (e.g. Pichia) as alpha-factor fusion protein that will be secreted into culture medium. After purification, the C-terminal glycine of the engineered polypeptide precursor can be converted to amide by enzymatic amidation, e.g. peptidylglycine alpha-amidating monooxygenase (PAM).
  • PAM monooxygenase
  • Peptides can be purified by any number of methods known in the art, including as described herein.
  • peptides are purified by RP-HPLC (preparative and analytical) using a Waters Delta Prep 3000 system.
  • a C4, C8 or Cl 8 preparative column (IOm, 2.2x25 cm; Vydac, Hesperia, Calif.) can be used to isolate peptides, and purity can be determined using a C4, C8 or C18 analytical column (5m, 0.46x25 cm; Vydac).
  • Amino acid analyses can be performed on the Waters Pico Tag system and processed using the Maxima program.
  • Peptides can be hydrolyzed by vapor-phase acid hydrolysis (115° C., 20-24 h). Hydrolysates can be derivatized and analyzed by standard methods (Cohen et al, THE PICO TAG METHOD: A MANUAL OF ADVANCED TECHNIQUES FOR AMINO ACID ANALYSIS, pp. 11-52, Millipore Corporation, Milford, Mass. (1989)).
  • Fast atom bombardment analysis can be carried out by M-Scan, Incorporated (West Chester, Pa.). Mass calibration can be performed using cesium iodide or cesium iodide/glycerol. Plasma desorption ionization analysis using time of flight detection can be carried out on an Applied Biosystems Bio-Ion 20 mass spectrometer.
  • Methods are available for assaying the level of protein expression by a host cell. Procedures useful for assaying the level of protein expression by a host cell are exemplified in the following typical protocol. About 25 pi BL21 E. coli cells are transformed with 2 ul plasmid DNA (expression vector for the engineered polynucleotide). Cells can be plated and incubated overnight at 37 degrees C. or at room temperature (RT) over a 48-hr period. A single colony can be selected and used to grow starter culture in 4 ml LB media with appropriate antibiotic for ' 6 hrs.
  • RT room temperature
  • Glycerol stocks can be prepared by adding 100 ul 80% sterile glycerol to 900 ul stock, which can then be mixed gently and stored at -80 C. A 250 m ⁇ sample can be removed for TCP uninduced sample. An aliquot, for example, 2 ml of Magic media containing appropriate antibiotic can be inoculated with 5 m ⁇ starter culture, which can then be incubated overnight (up to 24 hrs) at 37 C, 300 rpm. As known in the art, Magic Media is autoinducing.
  • 60 ml Magic Media containing appropriate antibiotic can be inoculated with 60 m ⁇ starter culture in a 250 ml or 125 ml Thompson flask, which can then be incubated overnight (up to 24 hrs) at 30 C, 300 rpm. After incubation, 250 pL culture can be removed from each tube and the cells pelleted. The cell can be resuspended in 1 ml 50 mM Tris pH 8, 150 mM NaCl, to which can be added 0.1 volumes (100 ul) POP culture reagent and 1 m ⁇ r-lysozyme (1:750 dilution in r-lysozyme buffer). The mixture can be mixed well and incubated at least 10 min at RT.
  • the preparation can then be centrifuge 10 min at 14000xG.
  • the supernatant (soluble fraction) can be removed and retained, and samples can be prepared for gel analysis (15 m1+5 m ⁇ LDS).
  • the remaining inclusion body pellet can be resuspended in 1 ml 1% SDS with sonication.
  • the sample can be prepared for gel analysis (15 ul+5 m ⁇ LDS).
  • 1.0 volumes POP culture reagent and 1 m ⁇ r- lysozyme (1:750 dilution in r-lysozyme buffer) can be added.
  • the mixture can be mixed well and incubated at least 10 min at RT. In embodiments, these samples do not need to be centrifuged.
  • the sample can then be prepared for gel analysis (15 m1+5 m ⁇ LDS).
  • NU-PAGE gels (4-12%) non-reduced in 1 xMES buffer can be run and stained with Simply Blue microwave protocol. Destaining can be conducted overnight, as known in the art. A gel image can be retained, and analyzed to determine protein expression levels.
  • the cell pellet can be resuspended in a minimum of 100 ml Lysis buffer for each 50 ml culture. Upon the addition of 30 ml, a 10 ml pipette can be used to resuspend, then the tube can be washed out with an additional 70 ml.
  • the resuspended cell solution can be multiply run, e.g., 4 passes, through a microfluidizer at 100 PSI (min) taking care to keep chamber in ice water through the entire process.
  • the fluidized slurry can be centrifuged at 14000xg, 20 min (e.g., JLA 10.5, 10,000 rpm).
  • the inclusion body pellet can be resuspended on ice in chilled lysis buffer with stir bar and stir plate for 1 hour at 4°C after disruption with pipette tip.
  • the pellet can be resuspended a second time in distilled EhO with stir bar and stir plate for 1 hour at 4°C after disruption with pipette tip, followed by centrifugation at 14000xg, 15 min.
  • the supernatant can be removed and discarded.
  • the resultant can be stored at -80°C.
  • Inclusion body pellets can be solubilized in appropriate volume of solubilization buffer (8M urea or 8M guanidine, 50 mM Tris, 10 mM DTT, pH 7.75) for 1 hour at RT.
  • solubilized pellets can be centrifuged for 20 min at 27 000 g.
  • Filtered (e.g., 0.4 um) supernatant can be transferred drop by drop into appropriate volume of refolding buffer (50 mM Tris-HCl, 1 M urea, 0.8 M arginine, 4 mM cysteine, 1 mM cystamine; pH 8) at RT.
  • Samples can be concentrated and run on a gel filtration column (SuperdexTM 75 26/60) at 1-2 ml/min in 4 C environment using a GE Healthsciences AKTAFPLCTM. Appropriate protein containing fractions can be identified via SDS-PAGE, pooled and run through a second gel filtration column. Pooled protein can then be concentrated in Amicon filter to appropriate concentration and assayed for endotoxin levels using, e.g., Endosafe® PTS Reader (Charles River), as known in the art.
  • a protein sample Once a protein sample has passed the endotoxin criteria, it can be sterile filtered, dispensed into aliquots and run through quality control assays.
  • Quality control assays can include analytical HPLC-SEC, non-reducing SDS PAGE and RP HPLC-MS to obtain approximate mass. Proteins can be obtained in 1 xPBS (137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM disodium phosphate, 1.4 mM monopotassium phosphate, pH7.2), distributed into aliquots and flash frozen for storage at -70 to -80° C.
  • a host cell e.g., a mammalian host cell
  • a histone polypeptide described herein can then be purified from the conditioned media using conventional techniques, such as immunoprecipitation, affinity chromatography, polypeptide A-sepharose, gel electrophoresis, and the like.
  • the pharmaceutical composition can comprise a therapeutic or diagnostic component and a pharmaceutically acceptable carrier, accipient or diluent.
  • compositions can comprise histone polypeptides or fragments thereof, or nucleic acids encoding the same, in combination with a pharmaceutically acceptable excipient (e.g., carrier).
  • a pharmaceutically acceptable excipient e.g., carrier
  • pharmaceutically acceptable carrier can refer to pharmaceutical excipients, for example, pharmaceutically, physiologically, acceptable organic or inorganic carrier substances suitable for administration to a subject that do not deleteriously react with the active agent.
  • Suitable pharmaceutically acceptable carriers include water, salt solutions (e.g., Ringer's solution and the like), alcohols, oils, gelatins, and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, and polyvinyl pyrrolidine.
  • Such preparations can be sterilized and, in embodiments, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
  • therapeutic agent or “therapeutic component” can refer to an agent or component that can induce a biological effect in vivo and/or in vitro.
  • the biological effect can be useful for treating and/or preventing a condition, disorder, or disease in a subject or patient.
  • the phrase “diagnostic component” can refer to a portion of a molecule that allows for the detection, imaging, and/or monitoring of the presence and/or progression of a condition, pathological disorder, and/or disease.
  • the therapeutic and/or diagnostic component can be an isolated or recombinant histone polypeptide or fragment thereof as described herein, a fusion protein as described herein, a cell as described herein, and/or a nucleic acid as described herein.
  • the pharmaceutical composition can further comprise at least one additional active agent.
  • additional active agent can refer to an agent useful alone, administered simultaneously, administered sequentially, or in combination with one or more additional agents, in the treatment, prophylaxis or palliative care of a subject afflicted with a disease or disorder.
  • the additional active agent can be employed in the compositions in an amount previously employed alone as a“standard of care”.
  • the additional active agent can be employed in the compositions in less than an amount previously employed alone as a“standard of care”.
  • the additional active agent can be co-administered or employed in a composition of the disclosure in an amount effective to cause measurable reduction of a symptom or sign of a disease, disorder or condition.
  • the additional active agent can include conventional cancer therapeutics such as chemotherapeutic agents, cytokines, chemokines, and radiation therapy.
  • chemotherapeutic drugs can he divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. These drugs can affect cell division or DMA synthesis and function in some way.
  • Additional therapeutics include monoclonal antibodies and the new tyrosme kinase inhibitors e.g. imatmib mesylate, which directly targets a molecular abnormality in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors).
  • chemotherapeutic agents include, but are not limited to cisplatin, carbopiatm, oxahpiatin, mechiorethamine, cyclophosphamide, chlorambucil, vincristine, vinblastine, vinorelbme, vindesine, taxol and derivatives thereof, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, epipodophyll otoxins, trastuzumab,titiuximab, and ntuximab, bevacizumab, and combinations thereof.
  • drug delivery platform can refer to a system to facilitate the prevention and/or treatment of a disease by functioning as the primary structural component (can be referred to as the “backbone”, “scaffold”, or “carrier”) for a functional moiety (such as an imaging moiety or a functional moiety).
  • the functional component can be loaded, infused, formed, fused to, conjugated to, or otherwise incorporated into the structural component (e.g., the histone polypeptide or fragment thereof).
  • the drug-deliver platform can comprise a fusion protein comprising a histone polypeptide or fragment thereof and at least one functional moiety.
  • the phrase “functional moiety” can refer to an element (i.e., component) that can perform a function, for example, an imaging function or a drug-delivery function.
  • functional moieties comprise a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof .
  • aspects of the invention are also drawn towards methods for delivery of a therapeutic and/or diagnostic molecule to a cell.
  • delivery to a cell or “cell delivery” can refer to introduction of therapeutic and/or diagnostic molecule as described herein (e.g., a polypeptide, fusion protein, or nucleic acid) into a cell, such as for therapeutic purposes.
  • aspects of the invention are drawn towards methods of transducing a cell with a therapeutic and/or diagnostic molecule.
  • the phrase “transducing a cell” or “cell transduction” can refer to the process of introducing a therapeutic and/or diagnostic molecule as described herein (e.g., a polypeptide, fusion protein, or nucleic acid) into a cell, such as for therapeutic purposes
  • embodiments comprise contacting a cell with an isolated or recombinant histone polypeptide or fragment thereof as described herein, wherein the polypeptide or fragment thereof is fused to a therapeutic and/or diagnostic component (e.g., cargo).
  • a therapeutic and/or diagnostic component e.g., cargo
  • the cell can be a cancer cell or the cell can be a non-cancer cell.
  • the cell can be a suspected of being a cancer cell.
  • cancer cell can refer to a cell undergoing early, intermediate or advanced stages of multi-step neoplastic progression, such as that previously described (Pitot et al., Fundamentals of Oncology, 15-28 (1978)). This includes cells in early, intermediate and advanced stages of neoplastic progression including “pre-neoplastic cells (i.e., “hyperplastic cells and dysplastic cells), and neoplastic cells in advanced stages of neoplastic progression of a dysplastic cell.
  • pre-neoplastic cells i.e., “hyperplastic cells and dysplastic cells
  • neoplastic cells in advanced stages of neoplastic progression of a dysplastic cell.
  • Cancer includes cells that are not metastatic or are metastatic.
  • the major types of cancer are carcinomas, sarcomas, melanomas, lymphoma, and leukemias.
  • Carcinomas originate in the skin, lungs, breasts, pancreas, and other organs and glands.
  • Lymphomas are cancers of lymphocytes.
  • Leukemia is cancer of the blood. It does not usually form solid tumors.
  • Sarcomas arise in bone, muscle, fat, blood vessels, cartilage, or other soft or connective tissues of the body.
  • Melanomas are cancers that arise in the cells that make the pigment in skin.
  • Non-limiting examples of cancers include ovarian cancer, breast cancer, lung cancer, prostate cancer, cervical cancer, pancreatic cancer, colon cancer, stomach cancer, esophagus cancer, mouth cancer, tongue cancer, gum cancer, skin cancer (e.g., melanoma, basal cell carcinoma, Kaposi's sarcoma, etc.), muscle cancer, heart cancer, liver cancer, bronchial cancer, cartilage cancer, bone cancer, testis cancer, kidney cancer, endometrium cancer, uterus cancer, bladder cancer, bone marrow cancer, lymphoma cancer, spleen cancer, thymus cancer, thyroid cancer, brain cancer, neuron cancer, mesothelioma, gall bladder cancer, ocular cancer (e.g., cancer of the cornea, cancer of uvea, cancer of the choroids, cancer of the macula, vitreous humor cancer, etc.), joint cancer (such as synovium cancer), glioblastoma, lymphoma, and leukemia.
  • ocular cancer
  • the cancer comprises one or more of a colon cancer, colorectal cancer, gastrointestinal cancer, breast cancer, bladder cancer, kidney cancer, leukemia, brain cancer, sarcoma, astrocytoma, acute myelogenous leukemia (AML), and diffuse large B-lymphoma.
  • a colon cancer colorectal cancer
  • gastrointestinal cancer breast cancer
  • bladder cancer kidney cancer
  • leukemia brain cancer
  • sarcoma astrocytoma
  • AML acute myelogenous leukemia
  • diffuse large B-lymphoma diffuse large B-lymphoma.
  • treat can refer to the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • the terms “subject” or “individual” or “animal” or “patient” or “mammal,” can refer to any subject for whom diagnosis, prognosis, or therapy is needed, as described herein.
  • Mammalian subjects can include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on.
  • the mammal is a
  • the term “disease” or “condition” can refer to a state of being or health status of a patient or subject that can be treated with the compounds or methods provided herein.
  • the disease or condition comprises cancer.
  • cancer can refer to cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including but not limited to solid tumors and lymphoid cancers, kidney, breast, lung, kidney, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, esophagus, and liver cancer, lymphoma, including but not limited to non-Hodgkins and Hodgkins lymphoma, leukemia, and multiple myeloma.
  • cancers which can include malignant primary or secondary tumors, include, but are not limited to, bone cancer, cancer of the larynx, gall bladder, rectum, head and neck, bronchi, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteosarcoma, Ewin's sarcoma, reticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hair-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuromas, cervical cancer, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, rhabdomyosarcoma, Kaposi's sarcoma, oteogenic and other
  • the methods comprise administering to the subject a pharmaceutical composition described herein, such as a pharmaceutical composition comprising an isolated or recombinant histone polypeptide or a fragment thereof.
  • the pharmaceutical composition can be administered in a therapeutically effective amount.”
  • therapeutically effective can refer to that amount of the composition sufficient to treat a disease and/or ameliorate one or more causes or symptoms of a disease or disorder. Amelioration, for example, only requires a reduction or alteration, not necessarily elimination.
  • therapeutically effective amount “therapeutic amount” and “pharmaceutically effective amount” are synonymous.
  • One of skill in the art can readily determine the proper therapeutic amount.
  • administration can refer to the act of physically delivering, e.g., via injection or an oral route, a substance as it exists outside the body into a patient, such as by oral, subcutaneous, mucosal, intradermal, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
  • administration of the substance can occur after the onset of the disease, disorder or condition or symptoms thereof.
  • Prophylactic treatment involves the administration of the substance at a time prior to the onset of the disease, disorder or condition or symptoms thereof.
  • one or more additional molecules can be administered (i.e., coadministered) to the subject.
  • co-administration can refer to simultaneous and sequential administration of two or more compounds or compositions.
  • An appropriate time course for sequential administration can be chosen by the physician, according to such factors as the nature of a patient's illness, and the patient's condition.
  • additional molecules that can be administered to the subject together with a composition as described herein can comprise conventional therapeutics, such as conventional cancer therapeutics.
  • cancer therapeutics include, but are not limited to, chemotherapeutic agents, cytokines, chemokines, and radiation therapy.
  • chemotherapeutic drugs can be divided into alkylating agents, antimetabolites, an thracy dines, plant alkaloids, topoisomerase inhibitors, and other anti-tumor agents. These drugs can affect cell division or DNA synthesis and function in some way.
  • Additional therapeutics include monoclonal antibodies and the new tyrosine kinase inhibitors e.g. imatimb mesylate, which directly targets a molecular abnormality in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors).
  • chemotherapeutic agents include, but are not limited to cisplatm, carboplatin, oxaliplatm, mechlorethamine, cyclophosphamide, chlorambucil, vincristine, vinblastine, vinorelbine, vindesme, taxoi and derivatives thereof, irmotecan, topotecan, amsacrine, etoposide, etoposide phosphate, temposide, epipodophyllotoxins, trastuzuinab (HERCEPT1N®), cetuximab, and rituximab, hevaeizumah, and combinations thereof.
  • H3 is a secreted protein
  • Brefeldin A-inhibitor of “classical” ER to Golgi Protein Secretion o inhibits Sec7-type-GTP-exchange factors thereby preventing coat protein assembly on ER to Golgi protein transport vesicles
  • A206K AAG (bold and underlined!
  • Histones are small, highly basic, nuclear proteins that serve as structural elements to condense DNA into chromatin and regulate its accessibility 1 .
  • histones released from dying cells can act as extracellular signaling molecules, without wishing to be bound by theory, all intracellular histone molecules have originated from the cell in which they reside 2 6 .
  • histone H3 is horizontally transferred between viable cells.
  • biotin ligase to detect secreted proteins 7 ⁇ 8 , we serendipitously discovered that histone H3 is selectively secreted by autophagic cells relative to histones H2A, H2B, and H4.
  • H3 secretion was confirmed using two independent H3 antibodies (FIG. 47, panel g) and in multiple cell lines including RCC4, UMRC2, 769-P, and A498 ccRCC lines, MCF7 and MDA-MB-231 human breast carcinoma cells, U20S human osteosarcoma cells, B16 mouse melanoma cells, and HOG human oligodendroglioma cells (FIG. 51, panels d-f).
  • RCC496 ccRCC lines MCF7 and MDA-MB-231 human breast carcinoma cells
  • U20S human osteosarcoma cells U20S human osteosarcoma cells
  • B16 mouse melanoma cells and HOG human oligodendroglioma cells
  • the secreted H3 is indicated to be monomeric, as determined by non-denaturing Native Gel Electrophoresis of spin column concentrated media (FIG. 51, panels g & h), and was sensitive to trypsin and proteinase K digestion, indicating that it was not encapsulated in an extracellular vesicle 14 (FIG. 51, panels I & j).
  • H2B-mEmerald RCC4 cells contained H3-HaloTag, as determined by single molecule imaging, with -80% of the protein in the cytoplasm and -20% in the nucleus (FIG. 56, panels c-g).
  • transfer of H4 was virtually undetectable. Without wishing to be bound by theory, uptake into the nucleus would increase upon dissolution of the nuclear membrane during mitosis. [00332]
  • the Cre expressing cells grew more slowly than the color switch cells, without wishing to be bound by theory, because targeting Cre recombinase to chromatin impairs cellular fitness.
  • the H3-Cre, but not H4-Cre, cells promoted recombination of the color switch reporter (FIG. 50, panels b-d, and FIG. 59, panel a).
  • H3-Cre-dependent recombination was also detectable when the Cre donor cells and color switch recipient cells were grown as separate tumors on opposite flanks (FIG. 50, panels e-h, and FIG. 59, panel b).
  • Histones released by dying cells can elicit danger signals by interacting with Toll-like receptors on cell surfaces and contribute to the pathogenesis of diseases such as septic shock 2,5 .
  • Our findings indicate that autophagy causes the specific secretion of H3 in a form that can cross cell membranes and enter the nuclei of other cells.
  • Our data indicate that certain PTMs are enriched or depleted on secreted H3 compared to the bulk H3 population, indicating that specific PTMs influence H3 secretion, uptake, or the information conveyed to recipient cells.
  • H3K14 acetylation appears to promote H3 secretion.
  • Solid tumors invariably contain hypoxic cells and therefore, without wishing to be bound by theory, secrete H3.
  • H3 contributes to any of the systemic manifestations of cancer, such as cachexia and hypercoagulability, and that they can serve as diagnostic or prognostic biomarkers.
  • Our findings that an oncohistone can cross cell membranes also indicate that certain oncohistones could play paracrine roles in transformation.
  • 786-0, 769-P, A498, U20S, MDA-MB-231, MCF7, and HEK293T cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) + 10 % Fetal Bovine Serum (FBS) + 1 % Penicillin/Streptomycin (Pen/Strep). RCC4 cells.
  • DMEM Modified Eagle’s Medium
  • FBS Fetal Bovine Serum
  • Pen/Strep Penicillin/Streptomycin
  • RCC4 cells UMRC-2 cells were maintained in DMEM + 10% FBS + 1% Pen/Strep.
  • B16-F10 mouse melanoma cells maintained in DMEM + 10% FBS + 1% Pen/Strep.
  • HOG cells human oligodendroglioma line
  • SF8628 maintained in DMEM + 10% FBS + 1% Pen/Strep. Tex.loxP.EG cells 2 were maintained in Roswell Park Memorial Institute (RPMI) Medium + 10%
  • 786-0 cells with doxycycline-inducible VHL expression have been previously descrived 3 and were maintained in DMEM + 10% FBS + 1 % P/S + 800 pg/mL G418.
  • DMEM + 10% FBS + 1 % P/S + 800 pg/mL G418 After lentiviral integration of pLX-304-BirA*-G3-ER (or empty vector control) 786-0 cells expressing doxycycline-inducible VHL and BirA*-G3-ER were maintained in DMEM + 10% FBS + 1 % P/S + 800 pg/mL G418 + 5 pg/mL blasticidin.
  • RCC4 stably expressing Tet-myr-GFP or expressing LC3-GFP were maintained in DMEM + 10% FBS + 1% Pen/Strep + 1 pg/mL puromycin.786-0 and RCC4 cells expressing the “color switch” Cre-reporter (pLV-CMV-LoxP-DsRed-LoxP-eGFP) were and maintained in DMEM + 10% FBS + 1% Pen/Strep + 1 pg/mL puromycin.
  • Biotin (Sigma- Aldrich) was prepared to a stock solution of 200 mM in autoclaved, milliQ filtered water and was diluted in cell culture medium to the indicated final concentration.
  • Doxy cy dine (Sigma- Aldrich) was prepared to a stock solution of 1 mg/mL in sterile phosphate-buffered saline (PBS) and was diluted in cell culture medium to the indicated final concentration.
  • PBS sterile phosphate-buffered saline
  • PI Propidium Iodide
  • Brefeldin A (Selleck Chemicals) was prepared as a stock solution of 10 pM in DMSO and was diluted in cell culture medium to the indicated final concentration.
  • Bafilomycin A1 (Cell Signaling Technologies) was prepared as a stock solution of 100 mM in DMSO and was diluted in cell culture medium to the indicated final concentration. Rapamycin was prepared as a stock solution of 1 mM in DMSO and was diluted in cell culture medium to the indicated final concentrations. Hoechst 33342 (ThermoFisher) was first diluted to a lOx stock (10 pg/mL in Opti-MEM) and then added to the cell culture medium to a final concentration of 1 pg/mL.
  • Plasmids [00347] Plasmids [00347] pCMV 6-H3 -FLAG and pCMV6-H3K27M-FLAG have been previously described 4 . mEmerald-H3-32 (Addgene #54115), mCherry-H3-23 (Addgene #55058), mEmerald-H4-23 (Addgene #54117) mCherry H4-23 (Addgene #55060), plix_403 (Addgene #41395), pLX304 (Addgene #25890), and 3xHA-TurboID-NLS_pCDNA3 (Addgene #107171), pLV -CMV -LoxP-DsRed-LoxP-eGFP (Addgene #65726), Tet-myr-GFP (Addgene #83468) pMRX-IP-GFP-LC3-RFP-LC3AG (Addgene #84
  • pLenti-CRISPR-V2-Hygro has been previously described and was cloned in the Kaelin Laboratory by swapping out the puromycin resistance gene in pLenti- CRISPR-V2 for a hygromycin resistance gene 5 .
  • Generation of pLenti-CRISPR-V2-Hygro with gRNA sequences targeting ATG7 and Beclin 1 (BECN1) are described in detail below.
  • pLenti-Efla-gate-PGK-Puro has been previously described 6 ⁇ 7 and the cloning of the histone fusion ORF cDNAs into pLenti-Efl a-gate-PGK-Puro and pbx_403, cloning of plx304- BirA*-G3-ER, and cloning of pLenti-Efla- BirA-NLS -PGK-Puro are all described in detail below.
  • pLenti-CRISPR-V2-Hygro was digested with BsmBI (New England Biolabs, no. R0580) or FastDigest Esp 31 (Life Technologies, no. FD0454) for 30 minutes (min) at 37°C, and the resulting linearized vectors were gel-purified.
  • Oligonucleotides encoding 4 independent sgRNAs targeting ATG7 or Becbn-1 from the Brunello sgRNA Library 8 with corresponding Bsm BI/Esp 31 overhangs added to facilitate ligation were synthesized by Integrated DNA Technologies . Oligonucleotides were annealed using 0.15 nmol of each sense and antisense oligonucleotides.
  • oligonucleotides were heated at 95°C for 4 min and allowed to slowly cool to room temperature. Annealed oligonucleotides were then diluted 1 : 100 in nuclease-free water and ligated into the linearized vectors using T4 ligase in a 4 hour (h) incubation at room temperature. A 2-pL aliquot of the ligation mixture was then transformed into 20 pL of HB101 chemically competent F coli cells (Promega, no. L2011).
  • Plasmids from ampicillin-resistant colonies were isolated by QIAprep Spin Plasmid Miniprep
  • sgRNA oligonucleotides used for editing are: sgATG7 #1 F: 5 ’C ACCGCCAGAAAATATTCCCCGGTG3 ’ sgATG7 #1 R: 5’AAACCACCGGGGAATATTTTCTGGC 3’ sgATG7 #2 F: 5’CACCGCTCTTGTAAATACCATCTGT3’ sgATG7 #2 R: 5’AAACACAGATGGTATTTACAAGAGC 3’ sgATG7 #3 F: 5’ C ACCGCTTGAAAGACTCGAGTGTGT3 ’ sgATG7 #3 R: 5’AAACACACACTCGAGTCTTTCAAGC 3’ sgATG7 #4 F: 5’ CACCGTCCTACTTTAGACTTGGACA3’ sgATG7 #4 R: 5’AAACTGTCCAAGTCTAAAGTA
  • the histone fusion ORF cDNA were cloned into pLenti-Efla-gate-PGK-Puro, for constitutive expression, or plix_403, for doxycycline-inducible expression, using the gateway cloning method 9 .
  • the histone fusion ORF cDNAs were amplified by PCR.
  • the histone3-mEmerald ORF cDNA was PCR amplified from mEmerald-H3-
  • the histone3-mCherry ORF cDNA was PCR amplified from mCherry-H3-23 (Addgene #55058) using the following primers (which include the attb sequences for gateway cloning): F: 5’
  • the histone4-mEmerald ORF cDNA was PCR amplified from mEmerald-H4- 23 (Addgene #54117) using the following primers (which include the attb sequences for gateway cloning): F:
  • the histone4-mCherry ORF cDNA was PCR amplified from mCherry H4-23 (Addgene #55060) using the following primers (which include the attb sequences for gateway cloning): F: 5’
  • H3-FLAG ORF was PCR amplified from pCMV6-H3.3 (Origene RC202257) using primers : F: 5’G
  • GGGGAC A AGTTTGTAC AAA AAAGC AGGCTTC ATGGCTCGTACAAAGCAGACTGCCC3 ’ and R: 5’ GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAAACCTTATCGTCGTCATCC3’
  • Each BP Clonase reaction consisted of 1 pL of the purified PCR product, 100 ng of pDONR-223 (at 100 ng/pL), 1 pL BP Clonase (ThermoFisher) and 2 pL of IX Tris-EDTA (TE) Buffer (pH 7), and was incubated for 2 h at room temperature.
  • H2A-mEmerald, H2A-mCherry, H2B-mEmerald, H2B-mCherry, H4- FLAG, H3-Cre, H4-Cre, H3-HaloTag and H4-HaloTag histone fusion ORF cDNA were synthesized as dsDNA gBlocks by Integrated DNA Technologies.
  • the synthetic dsDNA fragments contained a 5’ attbl site, a 3’ attb2 site, and introduced the same 23 amino acid linker used in the H3 and H4 mEmerald and mCherry fusions described above between the indicated histones and fusion partners.
  • each BP clonase reaction consisted of 10 pL of the gBlock resuspended to 10 ng/pL in IX TE, 100 ng of pDONR-223 (at 100 ng/pL), 1 pL BP Clonase (ThermoFisher). All BP Clonase reactions were incubated at room temperature for 2 hours, and then 2 pL of each clonase reaction was transformed into HB101 chemically competent E. coli. Successful transformants were selected by growth on Lysogeny Broth (LB)-agar supplemented with 50 pg/mL spectinomycin plates.
  • LB Lysogeny Broth
  • Drug- resistant colonies were picked and then grown overnight at 37 °C in 2 mL of LB + 100 pg/mL spectinomycin. Bacterial cultures were pelleted, and plasmid DNA was then isolated using Qiaprep Spin Miniprep Kit (Qiagen). The pDONR-223 vectors containing the histone fusion ORF cDNAs were verified by Sanger Sequencing using the M13F and M13R sequencing primers.
  • the histone fusion ORF cDNAs were then recombined from pDONR-223 into the lenti viral expression vectors pLenti-Efla-gate-PGK-Puro 3 ⁇ 5 ⁇ 6 or plix_403 (Addgene #41395) using LR Clonase (ThermoFisher).
  • Each LR Clonase recombination reaction consisted of 1 pL pDONR-223 containing the histone fusion ORF at 100 ng/pL.
  • 1 pL of the lentiviral expression vector at 100 ng/pL
  • 2 pL of LR clonase and 7 pL of IX TE Buffer (pH 7).
  • Point mutant Histone-Fusion ORF cDNAs (H3K14R, H3K14Q and H3K27M) were generated using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs).
  • Site-directed mutagenesis was performed on pCMV6-H3.3 and verified by Sanger Sequencing.
  • the mutant ORF cDNA was then recombined into a lentiviral expression vector using an LR recombination reaction (cloning methodology described above).
  • the final plasmid preps were confirmed using Sanger sequencing before generation of lentiviruses.
  • the primers used for site-directed mutagenesis were as follows: H3K14R-F: 5 CACCGGCGGTCGAGCGCCACGCA3’,
  • H3K14R-R 5 ’GATTTCCGAGCTGTCTGTTTAGTACGAGCC3 ’
  • H3K14Q-F 5’ CACCGGCGGTCAAGCGCCACGCA3’ H3K14R-R: 5’ GATTTCCGAGCTGTCTGTTTAGTACGAGCC ATG3 ’ .
  • H3K27M-F 5’ GGCTGCTCGCATGAGCGCGCCGG3 ’
  • H3K27M-R 5’ TTGGTAGCCAGCTGCTTGCGTGG3’.
  • BirA-ER cells were created by stably expressing the BirA*-G3-ER, a highly promiscuous and active variant of BirA fused to a KDEL tag for ER localization; this strategy has been previously successfully employed to localize BirA-G3* the the ER in vitro and in vivo 10 11 .
  • pLX-304-BirA-G3*-ER was cloned recombining the BirA-G3*-ER ORF cDNA 10 from a pDONR gateway entry clone into pLX304 using the gateway cloning methodology detailed above.
  • BirA-NLS cells were created by stably expressing the Turbo-ID variant of BirA fused to a SV-40 NLS signal and his strategy has been previously successfully employed to localize Turbo ID to the nucleus 11 .
  • the Turbo ID variant of BirA does not require a “target sequence” to biotinylate a substrate and has been extensively used in proximity ligation experimetns 12 .
  • HEK293FT Cells were plated at a density of 2.5 x 10 6 cells per 10-cm dish. 48 h later, they were transfected with 10 pg of lentiviral transfer vector, 8 pg of psPAX2, and 2 pg of pMD2.G. 72 h after transfection, the media was collected and filtered through a 45 pm filter, and stored at 4 °C. Fresh growth medium (DMEM + 10% FBS + 1% Pen/Strep) was then added to virus-producing HEK293FT cells.
  • DMEM + 10% FBS + 1% Pen/Strep was then added to virus-producing HEK293FT cells.
  • the media was again collected and filtered through a 45 pM filter, combined with the media collected at 72 h post transfection, and supplemented with polybrene at 8 pg/mL, before addition to the target cells for infection.
  • target cells to be infected were plated at a density of 1 x
  • Target cells were incubated with the virus containing media for 48 h at 37 °C. The virus- containing media was then removed, and fresh growth media was added for 24 h. Next, selective media was added: for infection with viruses derived from the pLenti-Efla-PGK- Puro backbone and for viruses from the plix_403 backbone 1 pg/mL puromycin was added, for infection with viruses derived from the pLX304 backbone 5 pg/mL blasticidin was added, and for CRISPR/Cas9 knockouts using viruses derived from the pLenti-CRISPR-V2-Hygro backbone 5 500 pg/mL hygromycin B was added.
  • HEK293FT cells were plated at a density of 2.5 x 10 6 cells per 10-cm dish. 48 h later, they were transfected with 10 pg of pMRX-IP-GFP-LC3-RFP-LC3AG (the retroviral transfer vector encoding GFP-LC3 and RFP-LC3AG) and 10 pg of pCL-Ampho (Novus Bio). Virus production and infection protocols were then identical to the lentiviral protocols detailed above. After infection, stable cells were generated by selection with lug/mL puromycin, and successful selection was confirmed by live-cell fluorescence microscopy visualizing GFP-LC3.
  • 786-0 cells were plated at a density of 4 x 10 6 cells per dish in 15-cm dishes in DMEM + 10% FBS + 1% Pen/Strep. 48 h later the growth media was removed, and the cells were washed twice with PBS prior to the addition of 15 mL of fresh Opti-MEM. 48 h later, the conditioned Opti-MEM media was collected, spun at lOOg for 5 min at 4 °C to gently pellet any cells that might have dislodged during collection, and transferred to a 3 kDa cut-off filter column made of regenerated cellulose (Millipore UFC900324). The column was spun at 3800g for 15 min at room temperature.
  • Trichloroacetic Acid (TCA) Precipitation of Conditioned Cell Culture Media
  • Cells were plated in 6-well culture dishes at a density of 2 x 10 5 cells per well. 48 h later, the cells were washed twice with PBS and 1.5 ml Opti-MEM was then added per well. After 48 h of culture in Opti-MEM, the media was collected and spun down at lOOg for 5 min at 4 °C to gently pellet any cells that might have dislodged during collection. After centrifugation, 1 mL of the conditioned media was transferred to a fresh tube. Next, 250 pL of 100% w/v TCA was added to conditioned media and the sample was incubated on ice for 30 min.
  • the samples were centrifuged at 17,000g for 15 min at 4 °C to pellet the precipitated protein, and the supernatant was removed.
  • the sample pellets were then washed three times with 800 pL acetone followed by a 5 min centrifugation at 17,000g and 4 °C. After removal of the final acetone wash, the pellets were air dried for 5 min at room temperature and then further dried for 5 min at 95 °C to drive off any residual acetone.
  • “Media” samples indicated on Immunoblot figures are TCA precipitated protein from 1 mL of conditioned Opti-MEM resuspended in 20 pL of lx Sample Buffer (unless otherwise indicated) and assayed by immunoblot along “Cells” samples (10 pg of whole cell lysate, prepared as indicated).
  • Cells were then resuspended in Cell Lysis Buffer (50 mM Tris, 250 mM NaCl, 1% Igepal, 0.1% SDS, 5 mM EDTA, 10 mM Na2P2Ch, 10 mM NaF), briefly vortexed, and incubated for 30 min on ice. The lysates were then clarified by centrifugation at 17,000g for 10 min at 4 °C and transferred to a new microcentrifuge tube. The protein concentration of the whole cell extracts was quantified using a BCA Protein Assay (Thermo Fisher Scientific, no. PI23227).
  • Membranes were blocked by incubation in 5% milk/tris-buffered saline (TBS)/0.1% Tween 20 (TBS-T) for 1 hour at room temperature, washed three times with TBS-T (5 min per wash), and then probed with primary antibody diluted in either TBS-T + 5% Bovine Serum Albumin (BSA) or TBS-T + 5% milk as indicated, and incubated overnight at 4 °C with gentle agitation.
  • TBS Tris-buffered saline
  • TBS-T TBSA
  • the primary antibodies used were: rabbit a-VHL (1:1000 in TBS-T + 5%
  • G3-ER a highly promiscuous variant of Turbo ID fused to a KDEL tag for ER localization 10 11 , or the corresponding empty vector, were plated at a density of 2 x 10 6 cells per dish in 15-cm dishes in DMEM + 10% FBS + 1% Pen/Strep + 5 pg/mL Blasticidin and 500 pg/mL G418, with or without 2 pg/mL doxy cy cline.
  • the cells were split and replated at a density of 4 x 10 6 cells per dish in 15-cm dishes in DMEM + 10% FBS + 1% Pen/Strep + 5 pg/mL Blasticidin and 500 pg/mL G418, with or without 2 pg/mL doxy cy cline.
  • the growth media was removed, and the cells were washed 2 times with PBS prior to the addition of 15 mL Opti-MEM without selective antibiotics, but supplemented with 20 pM biotin (with or without 2 pg/mL doxy cy cline).
  • the conditioned Opti-MEM media was collected, spun at lOOg for 5 min at room temperature to gently pellet any cells that might have dislodged during collection, and filtered through a 45 pm syringe filter.
  • the conditioned media was transferred to a 3 kDa cut-off filter column made of regenerated cellulose (Millipore UFC900324). The column was spun at 3800g for 15 min at room temperature. The flow through was then discarded and the column was spun for an additional 20 min, after which the remaining flow through was again discarded. The column was then washed twice with 10 mL of PBS per wash, each followed by centrifugation at 3800g for 15 min at room temperature and subsequent discarding of the flow through. The column retentate was removed by pipetting and kept for subsequent analysis. In pilot experiments these two PBS wash steps were found to be necessary to remove residual biotin that was retained and interfered with subsequent capture and detection of biotinylated proteins.
  • Electrophoresis was conducted in aXCell SureLock Mini-Cell Electrophoresis System (ThermoFisher) using Dark Blue Cathode Buffer containing 0.02 % G-250 (ThermoFisher) in the Upper (inner) Buffer Chamber and NativePAGE Running Buffer (Anonde Buffer) in the Lower (outer) chamber at 150 V for 30 min at 4 °C.
  • the Dark Blue Cathode Buffer (ThermoFisher) containing 0.02 % G-250 in the Upper Buffer Chamber was replaced with the Light Blue Cathode Buffer containing 0.002 % G-250 (ThermoFisher) and the electrophoresis was resumed at 150 V for approximately 2 h at 4 °C, at which point the dye front approached the end of the gel.
  • the PVDF membrane Prior to transfer, the PVDF membrane was incubated in 100% ethanol for 2 min and then incubated in TurboTransfer Buffer (BioRad) for 2 min. Proteins were transferred using the BioRad TurboTransfer system. After transfer, the PVDF membrane was destained by washing three times with 50% Methanol (10 min with gentle rocking per wash), once in TBST-T + 0.5% SDS (5 min with gentle rocking per wash), and three times in TBS-T (5 min with gentle rocking per wash). Membranes were blocked by incubation in 5% milk in TBS-T gently rocking for 1 hour at room temperature and immunoblotted as indicated. [00386] Protease Protection Assays
  • 786-0 cells were plated at a density of 4 x 10 6 cells per dish in 15-cm dishes in DMEM + 10% FBS + 1% Pen/Strep. 48 h later the growth media was removed, and the cells were washed twice with PBS prior to the addition of 15 mL of fresh Opti-MEM. 48 h later, the conditioned Opti-MEM media was collected, spun at lOOg for 5 min at room temperature to gently pellet any cells that might have dislodged during collection. Next, 1 mL of conditioned media was aliquoted per experimental condition.
  • Trypsin was added to a final concertation of 100 pg/ml, with or without 1% TritonX-100, and incubated for 30 min at 4 °C.
  • Proteinase K was added to a final concentration of 200 pg/mL, with or without 1% TritonX-100, and incubated for 1 h at 37 °C.
  • TCA was added to a final concentration of 25% w/v and the samples were processed using the TCA precipitation protocol described above.
  • PI Propidium Iodide
  • 786-0 cells were plated at a density of 5 x 10 4 cells per well in 12-well dishes in DMEM + 10% FBS + 1% Pen/Strep. 48 h later, the growth media was removed, and the cells were washed twice with PBS prior to the addition of 1.5 mL of fresh Opti-MEM, Opti- MEM + FBS, or OPTIMEM + the indicated concentration of puromycin per well. 48 h later, all attached and floating cells were collected and pelleted by centrifugation at lOOg for 5 min at 4 °C.
  • the pellets were then washed once with ice-cold PBS.
  • the cells were resuspended in ice cold FACS buffer (PBS + 1% BSA) supplemented with PI at a final concentration of 1 pg/mL.
  • PI PI uptake experiments
  • cells were prepared using the Dead Cell Apoptosis Kit with Annexin V FITC and PI for flow cytometry (ThermoFisher) per the manufacturer’s protocol.
  • U20S cells were plated at a density of 2 x 10 5 cells per well in 6-well dishes in DMEM + 10% FBS + 1% Pen/Strep. 24 h later, the growth media was removed, and the cells were washed twice with PBS prior to the addition of 1.5 mL of fresh Opti-MEM. 24 h later, the cells were transfected with the 1 pg Plasmid DNA of WT pCMV-H3-FLAG, empty vector control, or the indicated point mutant using 1 pL Lipofectamine 2000 per well. 24 h after transfection, the conditioned culture media was collected, TCA precipitated, and immunoblotted.
  • RCC4-mCherry, RCC4-H2B-GFP cells, 293T-H3-HaloTag and 293T-H4- HaloTag cells were cultured in the imaging media, which was prepared by supplementing FluoroBrite medium (Invitrogen) with 10% FBS, 1 mM glutamax and 0.1 mM nonessential amino acids.
  • H3-HaloTag and H4-HaloTag 293T “donor” cells were first washed three times with lx PBS and then cultured in Opti- MEM (Thermofisher) for 24 hours before adding O.lnM JF646-HTL for HaloTag labeling. After culture for 24h in JF646-HTL supplemented Opti-MEM, the conditioned media was harvested and centrifuged at l,500g for 10 mins.
  • Opti- MEM Thermofisher
  • 293T “donor” and RCC4 “recipient” cells were plated on ultra-clean cover glasses with a ratio of 1 : 10 in the imaging media. After culturing overnight, cells were washed with three times with PBS (5 min per wash) prior to being incubated with Opti-Mem for 48 hours. The labeling was performed in the imaging media with InM JF646-HTL for 30 mins.
  • the cells were washed 3 times (10 min per wash) with imaging media, and then 100 ng/ml Hoechst dye added to delineate cell nuclei. Then, the cover glasses were transferred to Attofluor metal holders (Thermofisher) and mounted onto the microscope for highly inclined and laminated optical sheet (HILO) imaging.
  • Attofluor metal holders Thermofisher
  • HILO highly inclined and laminated optical sheet
  • Proper emission filters (Semrock) were placed in front of the 3 cameras for simultaneous imaging of Hoechst, mCherry and JF646.
  • JF646 labelled H3-HaloTag and H4-HaloTag molecules were imaged at 50 Hz using a 642 nm laser with the excitation intensity of ⁇ 1000 W/cm 2 . 5000 frames were collected for each field of view.
  • the spot localization (x,y) was obtained through 2D Gaussian fitting based on MTT algorithms 13 .
  • the localization and tracking parameters are listed in the Table 1. Diffusion coefficients were calculated from tracks with at least 5 consecutive frames by the MSDanalyzer 14 with a minimal fitting R 2 of
  • 786-0 cells expressing H3-mEmerald, H3-mCherry, H4-mEmerald or H4- mCherry were plated at a density of 5 x 10 4 cells per “donor” (upper) well and 5 x 10 4 cells per “recipient” (lower) well in 24-well, 3 mM membrane transwell plates (MilliporeSigma CLS3398) in DMEM + 10% FBS + 1% Pen/Strep. 24 h later, the growth media was removed, and the cells were washed twice with PBS prior to the addition of 1.5 mL of fresh Opti-MEM per well.
  • Donor 786-0 cells expressing H3-FLAG were seeded at a density of of 4 x
  • the lysates were then clarified by centrifugation at 17,000g for 10 min at 4° C and transferred to new tubes. At this point, 20 pL of lysate was taken to be used as input.
  • magnetic streptavidin beads (ThermoFisher 65002) were first blocked by incubation in TBST containing 1% BSA with gentle rocking for 1 hour at room temperature. 30 pL of beads were added to 180 pi of “recipient” cell lysate and the samples were gently rocked for 1 hr at room temperature. The beads were washed twice with Cell Lysis Buffer, once with 2 M Urea, twice more with Cell Lysis Buffer, and then twice with TBS-T. After the final wash, the beads were resuspended in 20 pL of IX Sample Buffer and heated for 10 min at 95° C to elute the proteins.
  • the cells were counted and resuspended together at a density of 2.5 x 10 4 SF8628 cells per mL and 2.5 x 10 4 BirA-NLS expressing 786-0 cells per mL (a total density of 5 x 10 4 cells per mL). After thoroughly mixing by gentle vortexing (3 times for 5 seconds each) and subsequent inversion (3 times), the cells were plated by adding 1 mL of cell suspension per well in 12-well dishes. HOG cells which secrete H3, but do not express the H3K27M oncohistone, were identically co-cultured with BirA -NLS 786-0 cells as a control.
  • the growth media was removed and the cells were then washed twice with PBS before the addition of 1.5 ml of fresh Opti-MEM per well.
  • 24 h after adding the Opti- MEM media to the co-cultures 20 mM biotin was added.
  • the cells were washed 4 times with PBS, collected, detached with 0.05% trypsin for 5 min at 37 °C, resuspended in growth media, pelleted by centrifugation for 5 min at lOOg, washed twice more with PBS, and lysed with 200 pL Cell Lysis Buffer for 30 min on ice.
  • the lysates were then clarified by centrifugation at 17,000g for 10 min at 4° C and transferred to new tubes. At this point, 20 pL of lysate was taken to be used as input.
  • magnetic streptavidin beads (ThermoFisher 65002) were first blocked by incubation in TBST containing 1% BSA with gentle rocking for 1 hour at room temperature. 30 pL of beads were added to 180 pi of “recipient” cell lysate and the samples were gently rocked for 1 hr at room temperature. The beads were washed twice with Cell Lysis Buffer, once with 2 M Urea, twice more with Cell Lysis Buffer, and then twice with TBS-T. After the final wash, the beads were resuspended in 20 pL of IX Sample Buffer and heated for 10 min at 95° C to elute the proteins.
  • H3-Cre expressing RCC4 cells pLenti-Efla-H3-Cre-PGK-Puro
  • color switch Cre-reporter expressing RCC4 cells
  • Cells were then resuspended at a density of 2.5 x 10 4 H3-Cre expressing cells and of 2.5 x 10 4 “color switch” Cre-reporter cells per mL (a total density of 5 x 10 4 cells per mL). After thoroughly mixing by gentle vortexing (3 times for 5 second each) and inversion 3 times, cell were plated by adding 1 mL of cell suspension per well in 12-well dishes. 48 h later the growth media was removed, and the cells were washed twice with PBS prior to the addition of 1.5 mL/well of Opti-MEM.
  • Donor cells expressing H4-Cre were cocultured using identical protocols and included as controls.
  • Co-culture experiments for “donor” cells expressing histone 3 Cre fusion ORFS with the indicated point mutants (H3K14R, H3K14Q, and H3K27M in pLenti-Efla-H3-Cre-PGK-Puro) were carried out identically as described herein.
  • H3-Cre pLenti-Efla-H3-Cre-PGK-Puro expressing cells with tex.loxP.EG cells 2 (exhausted T-Cells which grow in suspension, harboring a lox-stop-lox GFP cassette) at a 1 : 1 ratio
  • H3-Cre expressing cells were plated at a density of 5 x 10 4 per well in 12-well dishes in DMEM + 10% FBS + 1% FBS + 1 pg/mL Puromycin.
  • Log-phase tex.loxP.EG cells were pelleted and resuspended in Opti-MEM at a density of 5 x 10 4 Cells/mL.
  • mice were injected subcutaneously into the flank of approximately 10-week-old female NCr mice (Taconic, #NCRNU-F) anesthetized by isoflurane inhalation.
  • H4-Cre 50/50 xenografts were prepared similarly. 10 mice were injected per experimental group. All procedures followed approved IACUC protocols, and mice were monitored daily for changes in weight, activity, and food and water intake. 5 weeks later, once palpable tumors had formed, the animals were sacrificed, and the tumor was processed for flow cytometry and immunoblot analysis.
  • the pellet was resuspended in 1 mL DMEM + 10% FBS + 1% Pen/Strep and passed through a 35 pm cell strainer a final time before flow cytometry analysis.
  • the cells were analyzed by live-cell flow cytometry at the Dana Farber Flow Cytometry Core, analyzing 20,000 cells per sample using a BD LSRFortessa flow cytometer with BD FACSDiva software. FCS files were exported and analyzed using FlowJo software.
  • Dual Flank Histone-Cre and Cre-Reporter Xenografts [00422] 786-0 Cells expressing H3-Cre or H4-Cre as well as 786-0 cells expressing the “color switch” Cre-reporter (pLV-CMV-LoxP-DsRed-LoxP-eGFP) were cultured to 90% confluence in DMEM+ 10% FBS + 1% P/S + 1 pg/ml Puromycin. After detaching the cells with 0.05% trypsin for 5 min at 37 °C, both the “donor” and “recipient” cells were resuspended to a density of 100 million cells/ml in in PBS + 0.2% FBS.
  • mice were injected subcutaneously.
  • mice For mouse fasting experiments food was removed from the cages of approximately 10-week-old Female Black 6 mice (Taconic, C57BL/6NTac) for 24 h. All procedures followed approved IACUC protocols, and the mice retained access to water, and were monitored for changes in activity and behavior. After 24 h of fasting, each mouse was put under deep isoflurane anesthesia, and up to 700 pL of whole blood was collected per animal by cardiac puncture, sacrificing the animal. Whole blood was transferred to a 1.5 mL microcentrifuge tube and incubated upright at room temperature for 30 min. The whole blood sample was then centrifuged at 1500g for 10 min at 4 °C.

Abstract

This invention is directed to isolated or recombinant histone polypeptides and fragments thereof, compositions comprising the same, and methods for using the same to deliver macromolecules to a cell.

Description

RECOMBINANT HISTONE POLYPEPTIDE AND USES THEREOF
[0001] This supplication is an International Application, which claims the benefit of priority from U.S. provisional patent application no. 63/212,071, filed on June 17, 2021, and U.S. provisional patent application no. 63/215,938, filed on June 28, 2021, the entire contents of each which are incorporated herein by reference in their entireties.
[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
GOVERNMENT INTERESTS
[0004] This invention was made with government support under Grant No. R35 CA210068 and Grant No. P50 CA101942 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD OF THE INVENTION
[0005] This invention is directed to isolated or recombinant histone polypeptides and fragments thereof, compositions comprising the same, and methods for using the same to deliver macromolecules to a cell. BACKGROUND OF THE INVENTION
[0006] A histone is a protein that provides structural support to a chromosome. In order for very long DNA molecules to fit into the cell nucleus, they wrap around complexes of histone proteins, giving the chromosome a more compact shape. Some variants of histones are associated with the regulation of gene expression.
SUMMARY OF THE INVENTION
[0007] An aspect of the invention is directed to an isolated or recombinant histone polypeptide comprising a peptide or fragment thereof having at least 90% identity to the amino acid sequence according to:
MARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQK STELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEACEAYLVGLFEDTNLCAIH AKRVTIMPKDIQLARRIRGER (SEQ ID NO: 1)
MARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQK STELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEACEAYLVGLFEDTNLCAIH AKRVTIMPKDIQLARRIRGERA (SEQ ID NO: 2)
MARTKQTARKSTGGKAPRKQLATKAARKSAPSTGGVKKPHRYRPGTVALREIRRYQK STELLIRKLPFQRLVREIAQDFKTDLRFQSAAIGALQEASEAYLVGLFEDTNLCAIH AKRVTIMPKDIQLARRIRGERA (SEQ ID NO: 3).
[0008] In embodiments, the isolated or recombinant histone polypeptide comprises or consists of the amino acid sequence according to SEQ ID NO: 1, 2, or 3. [0009] In embodiments, the isolated or recombinant histone polypeptide comprises one or more modifications. For example, the one or more modifications comprises one or more post- translational modifications and/or mutations. For example, the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3. For example, the mutation one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
[0010] In embodiments, the isolated or recombinant histone polypeptide fragment comprises an N-terminus truncated fragment or a C-terminus truncated fragment. In embodiments, the fragment consists of or comprises a fragment of SEQ ID NO: 1, 2 or 3, or a sequence at least 90% identical thereto.
[0011] In embodiments, the isolated or recombinant histone polypeptide exhibits at least one activity selected from the group consisting of crossing a cell membrane, secretion from a cell, uptake by a cell, or delivery of a macromolecule.
[0012] In embodiments, the isolated or recombinant histone polypeptide is a eukaryotic histone polypeptide.
[0013] In embodiments, the isolated or recombinant histone polypeptide is a mammalian histone polypeptide.
[0014] In embodiments, the isolated or recombinant histone polypeptide is isolated from a host cell which expresses the polypeptide. For example, the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell.
[0015] In embodiments, the isolated or recombinant histone polypeptide is encoded by a nucleic acid sequence according to 4, a fragment thereof, or a sequence at least 90% identical thereto.
[0016] In embodiments, the isolated or recombinant histone polypeptide comprises a fusion protein. [0017] In embodiments, the fusion protein comprises at least one functional moiety. For example, the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof. For example, the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin. For example, the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
[0018] In embodiments, the functional moiety comprises a macromolecule. For example, the macromolecule comprises a protein (such as an antibody, a cytokine, or a growth-inhibitor) or a chemical compound (such as a chemotherapeutic or toxin).
[0019] Aspects of the invention are also drawn to a nucleic acid encoding a histone polypeptide or fragment thereof according to SEQ ID NO: 1, 2, or 3, or an amino acid sequence having at least 90% identity thereto.
[0020] In embodiments, the nucleic acid comprises or consists of a sequence having at least 90% identity to SEQ ID NO: 4 or a fragment thereof. In embodiments the nucleic acid comprises or consists of SEQ ID NO: 4.
[0021] Aspects of the invention are also drawn to a cell comprising a nucleic acid encoding an isolated or recombinant histone polypeptide thereof. In embodiments, the cell is a mammalian cell, bacterial cell, yeast cell, or an insect cell.
[0022] Further, aspects of the invention are drawn to a fusion protein comprising an isolated or recombinant histone polypeptide comprising or consisting of an amino acid sequence of at least 90% identity to SEQ ID NO: 1, 2, or 3 or a fragment thereof and at least one functional moiety.
[0023] In embodiments, the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof. For example, the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin. For example, the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent. For example, the functional moiety comprises a macromolecule.
[0024] In embodiments, the macromolecule comprises a protein (such as an antibody, a cytokine, or a growth-inhibitor) or a chemical compound (such as a chemotherapeutic or a toxin).
[0025] In embodiments, the histone polypeptide comprises or consists of SEQ ID NO: 1, 2, or 3.
[0026] In embodiments, the histone polypeptide comprises one or more modifications. For example, the one or more modifications comprises one or more post-translational modifications and/or mutations. For example, the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3. For example, the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
[0027] Aspects of the invention are also drawn to a cell expressing an isolated or recombinant histone polypeptide thereof, or the fusion protein comprising or consisting of an isolated or recombinant histone polypeptide thereof.
[0028] In embodiments, the cell comprises an insect cell, a mammalian cell, a bacterial, or a yeast cell.
[0029] Still further, aspects of the invention are drawn to a composition comprising an isolated or recombinant histone polypeptide thereof, a fusion protein comprising or consisting of an isolated or recombinant histone polypeptide thereof, or a cell comprising the same, and a pharmaceutically acceptable carrier.
[0030] In embodiments, the composition further comprises at least one additional active agent. [0031] Aspects of the invention are also drawn towards a drug-delivery platform comprising an isolated or recombinant histone polypeptide comprising or consisting of an amino acid sequence of at least 90% identity to SEQ ID NO: 1, 2, or 3 or a fragment thereof.
[0032] In embodiments, the drug deliver platform comprises an isolated or recombinant histone polypeptide thereof comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, 2 or 3.
[0033] In embodiments, the isolated or recombinant histone polypeptide thereof of the drug- delivery platform comprises one or more modifications. For example, the one or more modifications comprises one or more post-translational modifications and/or mutations. For example, the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3. For example, the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
[0034] In embodiments, the drug-delivery platform comprises a fusion protein comprising an isolated or recombinant histone polypeptide thereof.
[0035] In embodiments, the fusion protein comprises at least one functional moiety. For example, the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof. For example, the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin. For example, the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
[0036] In embodiments, the functional moiety comprises a macromolecule. For example, the macromolecule comprises a protein (such as an antibody, a cytokine, or a growth-factor) or chemical compound (such as a chemotherapeutic or toxin). [0037] Aspects of the invention are further drawn to a method of delivering at least one functional moiety to a cell. For example, the cell can be a cancer cell, or the cell can be a non-cancer cell.
[0038] In embodiments, the method comprises contacting a cell with an isolated or recombinant histone polypeptide comprising the amino acid sequence of at least 90% identity to SEQ ID NO: 1, 2, or 3, or a fragment thereof, wherein the at least one functional moiety is fused to the histone polypeptide.
[0039] In embodiments, the isolated or recombinant histone polypeptide thereof comprises or consists of SEQ ID NO: 1, 2 or 3.
[0040] In embodiments, the histone polypeptide comprises one or more modifications. For example, the one or more modifications comprises one or more post-translational modifications and/or mutations. For example, the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3. For example, the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
[0041] In embodiments, the functional moiety can be a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof. For example, the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin. For example, the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent. [0042] In embodiments, the functional moiety comprises a macromolecule. For example, the macromolecule comprises a protein (such as an antibody, a cytokine, or a growth inhibitor) or chemical compound (such as a chemotherapeutic or toxin).
[0043] Aspects of the invention are also drawn towards a method of treating a subject afflicted with a disease, such as cancer. [0044] In embodiments, the method comprises administering to the subject an isolated or recombinant histone polypeptide comprising an amino acid sequence of at least 90% identity to SEQ ID NO: 1, 2, or 3 or a fragment thereof, wherein the histone polypeptide is fused to a functional moiety.
[0045] In embodiments, the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof. For example, the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin. For example, the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent. [0046] In embodiments, the functional moiety comprises a macromolecule. For example, the macromolecule comprises a protein (such as an antibody, a cytokine, or a growth inhibitor) or chemical compound (such as a chemotherapeutic or toxin).
[0047] In embodiments, the isolated or recombinant histone polypeptide thereof comprises or consists of SEQ ID NO: 1, 2, or 3.
[0048] In embodiments, the isolated or recombinant histone polypeptide thereof comprises one or more modifications. For example, the one or more modifications comprises one or more post-translational modifications and/or mutations. For example, the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3. For example, the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
[0049] Embodiments can further comprise administering to the subject an isolated or recombinant histone polypeptide fused to a functional moiety.
[0050] In embodiments, the isolated or recombinant histone polypeptide comprises histone H3. For example, histone H3 comprises histone H3.1, histone H3.2, or histone H3.3. [0051] In embodiments, the isolated or recombinant histone polypeptide comprises or consists of an amino acid sequence or fragment thereof according to SEQ ID NO: 1, 2 or 3, or a sequence at least 90% identical thereto.
[0052] In embodiments, the isolated or recombinant histone polypeptide comprises one or more modifications. For example, the one or more modifications comprises one or more post- translational modifications and/or mutations. For example, the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3. For example, the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
[0053] In embodiments, the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof. For example, the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin. For example, the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent. [0054] In embodiments, the functional moiety comprises a macromolecule. For example, the macromolecule comprises a protein (such as an antibody, a cytokine, or growth inhibitor) or chemical compound (such as a chemotherapeutic or toxin).
[0055] Aspects of the invention are also drawn towards a method of transducing a cell with a protein or chemical compound.
[0056] In embodiments, the method comprises contacting the cell with an isolated or recombinant histone polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1, 2, or 3 or a fragment thereof, wherein the histone polypeptide is fused to the protein or chemical compound. For example, the cell can be a cancer cell, or the cell can be a non-cancer cell.
[0057] In embodiments, the histone polypeptide comprises or consists of SEQ ID NO: 1, 2 or
3. [0058] In embodiments, the histone polypeptide comprises one or more modifications. For example, the one or more modifications comprises one or more post-translational modifications and/or mutations. For example, the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3. For example, the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
[0059] In embodiments, the protein comprises a therapeutic moiety or an imaging moiety. For example, the therapeutic moiety comprises a therapeutic protein (such as an antibody, a cytokine, or a growth inhibitor) or polypeptide, a small molecule (such as a chemical compound or chemotherapeutic), or a toxin. For example, the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
[0060] Other objects and advantages of this invention will become readily apparent from the ensuing description.
BRIEF DESCRIPTION OF THE FIGURES
[0061] FIG. 1 shows BirA is an E. coli biotin ligase that covalently attaches biotin to nearby proteins.
[0062] FIG. 2 shows methods of identifying secreted proteins using ER-targeted BirA. [0063] FIG. 3 shows 786-0 VHL Renal Cancer Cells Expressing ER-BirA and Dox- inducible VHL.
[0064] FIG. 4 shows detection of biotinylated secreted proteins.
[0065] FIG. 5 shows detection of extracellular H3 without the BirA system.
[0066] FIG. 6 shows posttranslational modifications on secreted H3 do not mirror bulk H3.
[0067] FIG. 7 shows H3K14 acetylation is highly enriched in secreted H3. [0068] FIG. 8 shows secretion of wildtype H3 and H3K14 mutants. The mutation of K to
R blocks acetylation, and the mutation of K to Q mimics acetylation.
[0069] FIG. 9A shows secretion of H3 is autophagy-dependent. Brefeldin A blocks canonical protein secretion, and Bafilomycin A1 blocks autophagy-dependent secretion. [0070] FIG. 9B shows secretion of H3 is autophagy-dependent using ATG7 knockout.
[0071] FIG. 9C shows secretion of H3 is autophagy-dependent using Beclin knockout.
[0072] FIG. 10 shows serum starvation promotes H3 secretion.
[0073] FIG. 11 shows autophagy inducer Rapamycin promotes H3 secretion.
[0074] FIG. 12 shows Dox-inducible H3 and H4 mEmerald and mCherry fusion proteins. [0075] FIG. 13 shows detection of dual H3-mEmerald and H3-mCherry positive cells in co-culture experiments.
[0076] FIG. 14 shows co-culture H3-cherry 293T cells with Myr (membrane)-GFP RCC4 cells resulting in rare green cells (myr-GFP) with red nuclear inclusions (H3-mcherry).
[0077] FIG. 15 shows inclusion corresponds to condensed chromosomes at metaphase plate.
[0078] FIG. 16 shows an experimental design of a transwell system. Pore size can be, for example, 0.2mM. Results show horizontal transfer of histone H3.
[0079] FIG. 17 shows “stamp upon receipt assay”, which demonstrates evidence of Histone H3 transfer using biotinylated H3-FLAG. Results show detection of H3 in the nucleus of recipient cells.
[0080] FIG. 18 shows single molecule imaging using HaloTag-H3 (or H4) 293T cells. For example, media is harvested from HaloTag-H3 (or H4) 293T cells and then added to mCherry RCC4 (Renal Cell Carcinoma) cells. Results show starvation selectively promotes H3 secretion into the culture medium, and also imaging uptake of Halo-Tag-Histone H3 into RCC cells. After incubation and transferring, 35 out of 217 RCC cells (16.1%) are positive for HaloTag signal.
[0081] FIG. 19 shows most of the H3 taken up by cells remains in the cytoplasm, but some gets to the nucleus.
[0082] FIG. 20 shows co-culture of rare HaloTag-Histone H3 293T donor cells and H4- mCherry RCC4 recipient cells.
[0083] FIG. 21 shows H3 transferred to nucleus and cytoplasm of recipient cells. Localization colored based on frame numbers.
[0084] FIG. 22 shows co-culture of Lox-Stop-Lox GFP reporter cells with H3-Cre expressing cells activates GFP expression.
[0085] FIG. 23 shows autophagy is not required for uptake of H3 (mCherry data).
[0086] FIG. 24 shows 50/50 tumors FACS data.
[0087] FIG. 25 shows co-culture with H3-Cre donor cells induces “color switch” recombination in recipient cells.
[0088] FIG. 26 shows “color switch” assay (Cre Reporter #1). See also, Zomeer et al. Nature Protocols, 2016.
[0089] FIG. 27 shows Histone H3-Cre fusion proteins induces “color switch” recombination in independent cells.
[0090] FIG. 28 shows H3 cytoplasmic fraction diffuses faster than the nuclear fraction. Median diffusion coefficient in the nucleus 0.3182 pm2/s. Median diffusion coefficient in the cytoplasm 0.8767 pm2/s. n=ll cells.
[0091] FIG. 29 shows GFP fluorescence in the “color switch” reporter assay is Cre- dependent.
[0092] FIG. 30 shows autophagy is not required for uptake of H3 (mEmerald data) [0093] FIG. 31 shows glycine substitution (removing AA side chain) at K4, K9, K27, or K36 does not decrease the levels of tagged-H3 in conditioned media.
[0094] FIG. 32 shows K to Q (acetylation mimic) at K27 and K36 decreases accumulation of the tagged-H3 in conditioned media.
[0095] FIG. 33 shows dectection of BirA-dependent, biotinylated extracellular proteins. [0096] FIG. 34 shows BirA-G3-ER system detects known, VHL-dependent differences in secreted peptides.
[0097] FIG. 35 shows no H3 is detected in unconditioned, concentrated media.
[0098] FIG. 36 shows detection of H3 secretion using orthogonal methodology (TCA precipitation) in multiple RCC cell lines using independent antibodies. Cells = 10pg of whole cell lysate; Media = TCA precipitated proteins from lmL serum-free OPTIMEM media after 48 hours in culture. IGFBP3 is known not to be secreted by 769P cells.
[0099] FIG. 37 shows H3 and H4 fluorescent fusion proteins have similar fluorescence intensity.
[00100] FIG. 38 shows 293FT H3-mCherry and RCC4 myr-GFP co-culture assay: single color controls.
[00101] FIG. 39 shows 293FT H3-mCherry and RCC4 myr-GFP co-culture shows some “dual positive” cells.
[00102] FIG. 40 shows forward and side scatter dot plots show cell-type enriched populations in RCC4/293FT co-culture assays.
[00103] FIG. 41 shows “dual positive” cells only appear in the RCC4-enriched population. [00104] FIG. 42 shows increasing the ratio of 293mCherry expressing cells increases the proportion of “dual positive” RCC4 cells.
[00105] FIG. 43 shows secreted H3 appears monomeric on native cells. [00106] FIG. 44 shows annotated nucleic acid and amino acid sequences of histone H3 fused to Cre recombinase.
[00107] FIG. 45 shows annotated nucleic acid and amino acid sequences of histone H3 fused to mEmerald.
[00108] FIG. 46 shows annotated nucleic acid and amino acid sequences of histone H3 fused to mCherry.
[00109] FIG. 47 shows (a) schema for protein biotinylation by BirA. (b-e). 786-0 VHL-/- renal carcinoma cells expressing BirA-ER or the corresponding empty vector (EV) were grown, where indicated, in the presence of 2 pg/mL doxy cy cline (Dox) for 4 days to induce exogenous VHL expression prior to the addition of 50 mM biotin. Cell lysates (Cells) and secreted proteins captured on streptavidin beads (Media) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with horseradish peroxidase-conjugated streptavidin (b) or with the indicated antibodies (c-e). (1) Immunoblot analysis of increasing amounts of 786-0 cell conditioned media after concentration with a spin-column or by TCA precipitation compared to whole cell extract and unconcentrated media (g-j) Immunoblot analysis of 786- O (g-i), RCC4 VHL-/- renal carcinoma (g-i), and U20S VHL+/+ osteosarcoma cell (j) extracts and TCA-precipitated conditioned media. In (j) the cells expressed the indicated FLAG-tagged histone H3 point-mutants.
[00110] FIG. 48 shows (a, f and g) immunoblot analysis of RCC4 cell extract and TCA precipitated conditioned media after the indicated treatments for 24 h. (b-e) Immunoblot analysis of RCC4 cells after CRISPR/Cas9-based editing with the indicated sgRNAs (NTC is Nontargeting Control). Cells = whole cell extracts. Media = TCA precipitated conditioned media (h and i) Immunoblot analysis of 786-0 cells grown in Opti-MEM synthetic media supplemented with 1% fetal bovine serum and the indicated amount of rapamycin for 24 h, or grown under the indicated oxygen concentration. Cells = whole cell extracts. Media = TCA precipitated conditioned media.
[00111] FIG. 49 shows (a and b). Data (a) and quantification (b) of 2 color FACS after co culturing 786-0 cells expressing the indicated histone fused to either mEmerald or mCherry (1 : 1 mix) in serum-free Opti-MEM for 2 days (c and d). Representative live cell fluorescence micrographs (c) and quantification (d) after coculturing 293T embryonic kidney cells expressing either H3 -mCherry or H4-mCherry with RCC4 cells expressing Myr-GFP in serum-free media for 2 days (e and 1). Data (e) and quantification (1) of 2 color FACS of recipient cells after growing the indicated donor and recipient 786-0 cells in transwell plates in serum-free media for 2 days (g) Immunoblot analysis of cell extract and TCA precipitated media from 786-0 cells expressing either H3-FLAG or H4-FLAG. (h) 786-0 cells expressing either BirA-NLS or EV were treated with conditioned media from 786-0 cells expressing either H3-FLAG or H4-FLAG for 2 days. Cell lysates before (input) and after (pull down) capture on streptavidin beads were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with horseradish peroxidase-conjugated streptavidin or with the indicated antibodies (i). Schematic for Cre recombinase reporter in Lox-Stop-Lox (L-S-L) GFP mouse T cells (j- k) Data (j) and quantification (k) after coculture of H3-Cre or H4-Cre 786-0 cells with L-S- L-GFP cells (1). Schematic for Color Switch Cre recombinase reporter in 786-0 cells (m-n) Data (m) and quantification (n) after coculture of H3-Cre or H4-Cre 786-0 cells with Color Switch reporter cells. For b,d,f,k,and n, data are mean ± s.e.m. from n = 3 biological replicates.
[00112] FIG. 50 shows (a and e). Schemas for in vivo transfer experiments in tumor bearing nude mice. Data (b) and quantification (c) of 2 color FACS of dissociated tumors formed 5 weeks after implanting 786-0 cells expressing either H3-Cre or H4 Cre mixed 1 : 1 with RCC4 color switch reporter cells (d) Immunoblot analysis of tumors from (a-c). 786-0 color switch reporter cells infected ex vivo with a Cre expressing lentivirus (+Cre) or the empty vector (EV) were included as controls (f and g) Data (1) and quantification (g) of 2 color FACS of tumors formed 786-0 color switch reporter cells 5 weeks after implanting them on one flank and 786-0 cells expressing either H3-Cre or H4 Cre on the opposite flank (h) Immunoblot analysis of tumors from (e-g). 786-0 color switch reporter cells infected ex vivo with a Cre expressing lentivirus (+Cre) or the empty vector (EV) were included as controls (i-j) Immunoblot analysis of liver lysates (i) and serum (j) from c57/B16 mice that were or were not fasted for 24 h. Each lane contains the same from a different mouse. For c and g, each dot represents an independent tumor, bars are mean ± s.e.m.
[00113] FIG. 51 shows (a) immunoblot analysis of cell extract from 786-0 cells expressing a doxycycline-inducible VHL. (b) 786-0 VEIL-/- renal carcinoma cells expressing BirA-ER or the corresponding empty vector (EV) were grown, where indicated, in the presence of doxycycline (Dox) for 4 days to induce exogenous VHL expression prior to the addition of 50 mM biotin. Cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with horseradish peroxidase-conjugated streptavidin or immunoblotted with the indicated antibodies (c) Immunoblot analysis of 786-0 cell extracts (Cells) and increasing amounts of 786-0 cell conditioned or unconditioned media after concentration with a spin- column. (d) Immunoblot analysis of 786-0, RCC4, UMRC2, 769P, and A498 cell extracts and TCA-precipitated conditioned media (e) Immunoblot analysis of 293T, U20S, B16-F10, HT1080, MDA-MB-231, and MCF7 cell extracts and TCA-precipitated conditioned media.
(1) Immunoblot analysis of 786-0, Human Oligodendroglioma (HOG) and SF8628 (H3F3A K27M) cell extracts and TCA-precipitated conditioned media (g-h) Immunoblot analysis of non-denaturing, native gel electrophoresis of 786-0 cell conditioned media after concentration with a spin-column, recombinant nucleosomes (g), and recombinant monomeric H3 (h). (i and j) Immunoblot analysis of TCA precipitated 786-0 cell conditioned media after treatment with 100 mg/mL Proteinase K for 1 h at 37 °C (i) or 100 pg/mL trypsin for 30 min at 4 °C (j). (k) Immunoblot U20S osteosarcoma cell extracts and TCA- precipitated conditioned media after transfection with the indicated FLAG-tagged histone H3 point-mutants.
[00114] FIG. 52 shows (a, b and c) data (a) and quantification (b,c) of FACS analysis of Propidium Iodide uptake of 786-0 cells that had been grown in Opti-MEM, Opti-MEM + 1% FBS, or Opti-MEM + indicated dose of Puromycin for 48 hours (d and e) Data (d) and quantification (e) of 2 color FACS analysis of Propidium Iodide uptake and Annexin V staining of 786-0 cells that had been grown in Opti-MEM, Opti-MEM + 1% FBS, or Opti- MEM + indicated dose of Puromycin for 48 hours. (1) Immunoblot analysis of 786-0 cells that had been grown in Opti-MEM, Opti-MEM + 1% FBS, or Opti-MEM + indicated dose of Puromycin for 48 hours (g) Immunoblot analysis of whole cell extracts (Cells) and TCA precipitated conditioned media (Media) from 786-0 cells that had been grown in Opti-MEM, Opti-MEM + 1% FBS, or Opti-MEM + the indicated concentration of Puromycin for 48 hours. For a-g, Puromycin was used as a positive control to induce cell death (h) Representative live-cell fluorescence micrographs of RCC4 cells expressing GFP-LC3 that had been grown in the indicated media for 48 hours (i) 786-0 VEIL-/- renal carcinoma cells expressing BirA-ER or the corresponding empty vector (EV) were grown in the presence of 50 mM biotin. Cell lysates (Cells) and secreted proteins captured on streptavidin beads (Media) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with horseradish peroxidase-conjugated streptavidin or with the indicated antibodies (j) Immunoblot analysis of TCA precipitated 786-0 conditioned media after the indicated treatments for 24 h.
[00115] FIG. 53 shows (a) representative live-cell fluorescence micrographs of 786-0 cells constitutively expressing H2A, H2B, H3, or H4 fused to either mEmerald or mCherry. (b) Representative live cell fluorescence micrographs of 786-0 cells expressing doxycycline- inducible H3 or H4 fused to either mEmerald or mCherry that were grown in media that did (+Dox) or did not (-Dox) contain lpg/mL Doxy cy cline for 4 days (c and d) Data (c) and quantification (d) of 2 color FACS after coculturing 786-0 cells expressing the indicated doxycycline-inducible H3 or H4 fused to either mEmerald or mCherry (1 : 1 mix) in serum- free Opti-MEM that did or did not contain 1 pg/mL doxy cy cline for 2 days (e and f) Data (e) and quantification (f) of 2 color FACS after coculturing 293T cells expressing doxycycline- inducible H3 or H4 fused to either mEmerald or mCherry (1 : 1 mix) in serum-free Opti-MEM that did or did not contain 1 pg/mL doxy cy cline for 2 days.
[00116] FIG. 54 shows (a and b) data (a) and quantification (b) of 2 color FACS after co culturing RCC4 cells expressing the indicated histone fused to either mEmerald or mCherry (1:1 mix) in serum-free Opti-MEM or Opti-MEM+ 1% FBS at the indicated oxygen concentration for 2 days.
[00117] FIG. 55 shows (a and b) data (a) and quantification (b) of FACS analysis of recipient RCC4 cells that had first undergone CRISPR/Cas9 editing with indicated guide RNAs and then were grown in conditioned medium from 293T cells expressing either H3 or H4 fused to mEmerald for 48 hours (c and d) Data (c) and quantification (d) of FACS analysis of recipient RCC4 cells that had first undergone CRISPR/Cas9 editing with indicated guide RNAs, after 48h in culture with conditioned medium from 293T cells expressing either H3 or H4 fused to mCherry. (e and f) 786-0 cells expressing either BirA-NLS or EV were co-cultured with SF8628 (H3K27M) or HOG (WT H3) cells for 2 days. Cell lysates before (input) and after (pull down) capture on streptavidin beads were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with horseradish peroxidase-conjugated streptavidin or with the indicated antibodies [00118] FIG. 56 shows (a and b) Representative Total Internal Reflection fluorescence (TIRF) microscopy Images (a) and quantification of single molecule localizations (b) of conditioned DMEM + 10% FBS (No Starvation) or Opti-MEM (Starvation) media from H3- HaloTag or H4-HaloTag expressing 293T cells labeled with JF646-HTL. Scale bar, 10 pm.
In the box chart, top and bottom error bars represent 95th and 5th percentiles, respectively; box represents the range from the 25th to 75th percentile; center line represents the median and the small square represents mean value (***, p value < 0.01, paired sample T test) (c-d) HILO single molecule localization microscopy of H3 -Halo tag molecules transferred into recipient RCC4 cells after 24 h culture in conditioned Opti-MEM from H3-HaloTag expressing 293T cells. The reconstructed images consist of localizations color coded by frame numbers (c) and local densities (d). The nucleus is highlighted by the dotted circle. Scale bar, 10 pm. (e) Quantification of H3-Halo or H4-Halo positive RCC4 cells recipient cells after co-culture with 293T H3-HaloTag or H4-HaloTag donor cells for 48 hours in Opti-MEM. (***, p value < 0.01, paired sample T test) (f) Quantification of the nuclear and cytoplasmic fractions of H3-HaloTag molecules detected in RCC4 recipient cells after co-culture with 293T H3- HaloTag or H4-HaloTag donor cells for 48 hours in Opti-MEM. The dotted line indicates the mean (g) Density histogram of diffusion coefficients for the cytoplasmic and nuclear H3- HaloTag fractions showed in (f).
[00119] FIG. 57 shows (a) immunoblot analysis of cell extracts and 3 mL of TCA precipitated conditioned media from 786-0 cells expressing the indicated H3 point mutants fused to Cre Recombinase (Cre) after 48 h culture in serum-free Opti-MEM. (b and c) Data (b) and quantification (c) of FACS analysis 24 h after co-transfecting the expression plasmids for histone Cre fusions along with a LoxP-STOP-LoxP-GFP reporter plasmid (d and e) Data (d) and quantification (e) after coculture of 786-0 cells expressing the indicated H3 point mutants fused to Cre with Color Switch reporter cells (1:1 mix) in serum-free Opti-MEM for 2 days.
[00120] FIG. 58 shows (a) representative live cell fluorescence micrographs of 786-0 cells expressing the indicated H3 point mutants fused to either mEmerald or mCherry. (b and c) Data (b) and quantification (c) of FACS after coculturing 786-0 cells expressing the indicated H3 point mutants fused to mCherry with RCC4 cells expressing myr-GFP (1:1 mix) in serum-free Opti-MEM + lpg/mL doxy cy cline for 2 days (d and e). Data (d) and quantification (e) of 2 color FACS after coculturing 786-0 cells expressing the indicated doxycycline-inducible H3 point mutants fused to either mEmerald or mCherry (1 : 1 mix) in serum-free Opti-MEM, in the presence of + lpg/mL doxy cy cline (Dox) where indicated, for 2 days. The data for the cells grown without Dox is not shown due to space.
[00121] FIG. 59 shows (a) immunoblot analysis of the second cohort of tumors formed 5 weeks after implanting 786-0 cells expressing either H3-Cre or H4 Cre mixed 1:1 with RCC4 color switch reporter cells (b) Immunoblot analysis of tumors from the second cohort of 786-0 color switch reporter cells 5 weeks after implanting them on one flank and implanting 786-0 cells expressing either H3-Cre or H4 Cre on the opposite flank.
DETAILED DESCRIPTION OF THE INVENTION [00122] Previous protein transduction methods have not been sufficiently robust for clinical use (e.g. protein transduction domains derived from HIV TAT or the homeodomain of Antennapedia). Delivering a protein to cells in the clinic currently requires genetic approaches (e.g. viral-based gene therapy, mRNA-based vaccines).
[00123] As described herein, it was discovered that cells naturally secrete histone proteins (e.g., histone H3) in response to autophagy and that this secreted form of histone H3 can cross cell membranes to deliver proteins to which they are fused both in cell culture and in mice. Importantly, cell uptake does not require autophagy. Further, it was discovered that the secreted histone H3 has specific posttranslational modifications that increase or decrease secretion and, without wishing to be bound by theory, transduction. Thus, aspects of the invention can be used to systemically deliver large molecules, such as proteins, that have been designed to modulate intracellular targets or replace intracellular targets.
[00124]
[00125] Abbreviations and Definitions
[00126] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the invention can be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the invention in any appropriate manner.
[00127] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” [00128] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.
[00129] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.
[00130] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.
[00131] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).
[00132]
[00133] Recombinant or Isolated Polypeptide
[00134] Aspects of the invention are drawn to an isolated or recombinant histone polypeptide.
[00135] The term “histone” can refer to DNA binding structural proteins of chromosomes. Histones have a high proportion of positively charged amino acids such as lysine and arginine, which aids in DNA binding.
[00136] The five main types of histones fall into two groups: nucleosomal histones H2A, H2B, H3, H4; and HI histones. The families H2A, H2B, H3, and H4 constitute the core histones, while the family of H1/H5 histones is known as the linker histones. Each nucleosome core comprises 8 histones, two of each of the core histones, around which the DNA winds. The linker histones thereby sit on top keeping the DNA in place and linking the nucleosomes to form higher-order structures. [00137] The core histones are highly conserved across eukaryotes in terms of sequence and structure. The skilled artisan will recognize that embodiments described herein can comprise histone polypeptides or fragments thereof from one or more eukaryotic species. For example, embodiments can comprise a histone polypeptide or fragment thereof corresponding to a mammalian histone.
[00138] The term '“Histone H2A” can refer to to a variety of closely related proteins that vary' often by only a few amino acids. These can be further classified by the subfamilies Histone H2A F, 112 A 1 , and H2A 2 and include the specific histones H2AFBL H2AFB2, H2AFB3, H2AFJ, H2AFV, 11.2 AFX. H2AFY, H2AFY2, H2AFZ, HIST1H2AA, HTST1H2AB, H1ST1H2AC, HiST]H2AD, HIST1H2AE, HIST1H2AG, HTST1H2AT, H1ST1H2AJ, HIST1H2AK, HIST1H2AL, HIST1H2AM, H1ST2H2AA3, and HIST2H2AC. H2A consists of a main globular domain and a long N-terminal tail or C -terminal on one end of the molecule. Hie N-terminal tail or C -terminal tail is the common location of post- translational modification.
[00139] The term ‘Histone H2FT also refers to a variety of closely related proteins further classified by the subfamilies H2BF, H2B1, and H2B2 and includes the specific exemplary histone proteins H2BFM, H2BFS, H2BFWT, H1ST1H2BA, HIST1H2BB, HIST1H2BC, HIST1H2BD, HIST1H2BE, HIST1H2BF, HIST1H2BG, HIST1H2BH, H1ST1H2B1, H1ST1H2BJ, BIST1 B2BK, F1IST1F12BL, BIST1 B2BM, HIST1H2BN, BIST1B2BO, and F1IST2F12BE.
[00140] The term “Histone B3” refers to one of the fi ve main families of histone proteins and represents the most extensively modified of the histone proteins by post-translational modifications. Specific subfamilies of histone B3 include H3A1, H3A2, and B3A3 and include the exemplary B3 histones FIIST1FI3A, HIST1H3B, HIST1H3C, HIST1H3D,
FITST1FI3E, FIIST1FI3F, HI5T1H3G, ITIST1IT3FT FIIST1FI3I. FITST1FI3J, TdIST2Td3C, and HIST3H3. As the most modified of the histone proteins, 113 histones have emerged to play an important role in gene regulation and the emerging science of epigenetics.
[00141] Featuring a main globular domain and a long N terminal tail, the “Histone H4” family comprises the families H41 and H44 histones and includes the exemplary histones HXST1H4A, HIST1H4B, HTST1H4C, HTST1H4D, HIST1H4E, HIST1H4F, ! HS ! ! f !4G. HTST1H4H, HIST1H4I, HIST1H4I HTST1H4K, HISTIH4L, and HTST4H4.
[00142] In embodiments, the histone can be a mammalian histone, polypeptide thereof, or fragment thereof. The terms “polypeptide,'” “peptide” and “protein” can be used interchangeably herein and can refer to a polymer of amino acid residues. The terms can apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring ammo acid, as well as to naturally occurring ammo acid polymers and non-naturally occurring ammo acid polymer.
[00143] The phrase “amino acid” and “amino acid residue” can refer to natural amino acids, unnatural amino acids, and modified amino acids. Any reference to an amino acid, generally or specifically by name, includes reference to both the D and the L stereoisomers if their structure allows such stereoisomeric forms. Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). Unnatural amino acids include, but are not limited to homolysine, homoarginine, homoserine, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4- aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3- aminoisbutyric acid, 2-aminopimelic acid, tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N- ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3 -hydroxy proline, 4- hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N- methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline, norleucine, ornithine, pentylglycine, pipecolic acid and thioproline. Additional unnatural amino acids include modified amino acid residues which are chemically blocked, reversibly or irreversibly, or chemically modified on their N-terminal amino group or their side chain groups, as for example, N-methylated D and L amino acids or residues wherein the side chain functional groups are chemically modified to another functional group. For example, modified amino acids include methionine sulfoxide; methionine sulfone; aspartic acid-(beta- methyl ester), a modified amino acid of aspartic acid; N-ethylglycine, a modified amino acid of glycine; or alanine carboxamide, a modified amino acid of alanine Additional residues that can be incorporated are described in Sandberg et al., J. Med. Chem. 41: 2481-91, 1998. [00144] Amino acid analogs can refer to compounds that have the same basic chemical structure as a naturally occurring ammo acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an ammo group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfomum. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Ammo acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring ammo acid.
[00145] The term “polypeptide” can encompass a singular “polypeptide” as well as plural “polypeptides,” and can refer to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” can refer to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specific nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
[00146] A polypeptide of the invention can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three- dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.
[00147] With regard to the amino acid sequence, histone H3 includes the proteins of Swiss Prot AccNo. Q93081 (Histone H3), P68431 (Histone H3.1), Q71D13 (Histone H3.2), and P84243 (Histone H3.3) (each of which are incorporated by reference herein in their entireties with respect to the sequence itself) and variants thereof, including, but not limited to, the modified histone proteins thereof, as well as proteins which are substantially identical thereto, and, optionally, also lack the N-terminal methionine residue at position 1 of the sequences provided herein (e.g., a post-translational loss of the N-terminal methionine residue). In embodiments, the histone polypeptide can comprise an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identical to Swiss Prot Acc No. Q93081 (Histone H3), P68431 (Histone H3.1), Q7D13 (Histone H3.2), or P84243 (Histone H3.3).
[00148] In embodiments, the histone polypeptide comprises an amino acid sequence according to (Start codon indicated by M - Met.):
Figure imgf000028_0001
[00149] The terms “identical” or “percent identity,” in the context of two or more nucleic acids or polypeptide sequences, can refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a specified region). Percent identity can be measured, for example, using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters set, or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” The definition can also include sequences that have deletions and/or additions, as well as those that have substitutions. Algorithms can account for gaps and the like.
[00150] For sequence comparison, for example, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are selected, if necessary, and sequence algorithm program parameters are selected. Default program parameters can be used, or alternative parameters can be selected. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
[00151] In embodiments, the histone polypeptide or fragment thereof can be a recombinant polypeptide. The term “recombinant” can refer to genetic material (i.e., nucleic acids, the polypeptides they encode, and vectors and cells comprising such polynucleotides) that has been modified to alter its sequence or expression characteristics, such as by mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence to that of another gene, placing a gene under the control of a different promoter, expressing a gene in a heterologous organism, expressing a gene at a decreased or elevated levels, expressing a gene conditionally or constitutively in manner different from its natural expression profile, and the like. Recombinant nucleic acids, polypeptides, and cells based thereon, have been manipulated by man such that they are not identical to related nucleic acids, polypeptides, and cells found in nature. The term “recombinant polypeptide” can refer to a polypeptide that is produced by recombinant techniques, wherein DNA or RNA encoding the expressed protein is inserted into a suitable expression vector that is in turn used to transform a host cell to produce the polypeptide.
[00152] Routine methods for making recombinant nucleic acids can be used to construct expression vectors encoding the polypeptides of interest using appropriate transcriptional/translational control signals and the protein coding sequences. (See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d Ed. (Cold Spring Harbor Laboratory 2001)).
[00153] In embodiments, the histone polypeptide or fragment thereof can be referred to as an “isolated polypeptide”. The term “isolated” can refer to a molecule of interest (such as, for example, a polynucleotide or a polypeptide) that is in an environment different from that in which the molecule can naturally occur. For example, an “isolated” molecule is one which is substantially separated from the cellular components (e.g., membrane lipids, chromosomes, proteins) of the host cell from which it originated, or from the medium in which the host cell was cultured. The term does not require that the biomolecule has been separated from all other chemicals or molecules, although certain isolated biomolecules can be purified to near homogeneity. For example, an “isolated polypeptide” can refer to a polypeptide that is free from at least one contaminating polypeptide or other contaminants that are found in its natural environment. In embodiments, the isolated polypeptide is substantially free from any other contaminating polypeptides or other contaminants that are found in its natural environment which can interfere with its therapeutic, diagnostic, prophylactic or research use. [00154] In embodiments, the histone polypeptide can comprise one or more modifications. The term “modification” or “protein modification” can refer to one or more post-translational modifications or amino acid mutations (e.g., substitutions, deletions, and/or insertions), as is well understood in the art.
[00155] The term “amino acid mutation” can refer to one or more mutations of amino acid positions on a fragment of polypeptide and variants thereof, wherein the variant can be obtained by substituting, inserting or deleting amino acids at one or some sites on the polypeptide. For example, an amino acid mutation of the histone H3 polypeptide can comprise a substitution of Lys-14 with Gln-14 according to SEQ ID NO: 1, 2, or 3, such as to increase histone secretion and transduction. Substituting Lys-14 with Arg-14 prevents secretion and transduction, whereas changing Lys-14 to Gln-14 increases secretion and transduction. Other non-limiting examples of amino acid mutations include a substitution of Lys-9 to Met-9 according to SEQ ID NO: 1, 2, or 3; Lys-14 to Gln-14 according to SEQ ID NO: 1, 2, or 3; Lys-27 to Met-27 according to SEQ ID NO: 1, 2, or 3; or any combination thereof. For example, the combination of amino acid mutations can comprise two or more substitutions, such as a substitution of Lys-14 to Gln-14, Lys-9 to Met-9, and Lys-27 to Met- 27. For example, substitution of Lys-14 to Gln-14 increases transfer. For example, substitution Lys-27 with Met-27 increases both secretion and transduction.
Figure imgf000031_0001
[00156] The phrase “post-translational modifications” can refer to one or more modifications that occur on a peptide after its translation by ribosomes is complete.
A post-translational modification can be a covalent modification and/or enzymatic modification. In embodiments, the covalent modification can be catalyzed by an enzyme. Examples of post-translation modifications include, but are not limited to, acylation, acetylation, alkylation (including methylation), biotinylation, butyrylation, carbamylation, carbonylation, deamidation, deiminiation, diphthamide formation, disulfide bridge formation, eliminylation, flavin attachment, formylation, gamma-carboxylation, glutamylation, glycylation, glycosylation, glypiation, heme C attachment, hydroxylation, hypusine formation, iodination, isoprenylation, lipidation, lipoylation, malonylation, methylation, myristolylation, oxidation, palmitoylation, pegylation, phosphopantetheinylation, phosphorylation, prenylation, propionylation, retinylidene Schiff base formation, S- glutathionylation, S-nitrosylation, S-sulfenylation, selenation, succinylation, sulfmation, ubiquitination, and C-terminal amidation. A post-translational modification can include modifications of the amino terminus and/or the carboxyl terminus of a peptide. Modifications of the terminal amino group include, but are not limited to, des-amino, N-lower alkyl, N-di- lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., wherein lower alkyl is C1-C4 alkyl). A post- translational modification also includes modifications, such as but not limited to those described herein, of amino acids falling between the amino and carboxy termini. The term post-translational modification can also include peptide modifications that include one or more detectable labels.
[00157] Modified histone proteins are well known in the art. For instance, the suitable histone protein modifications on histone H3.1 can include, but are not limited to, any one or more listed herein. For instance, the suitable histone protein modifications on a histone protein according to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a fragment or variant thereof, can include, but are not limited to, any one or more listed herein. Exemplary protein modifications for use according to the invention are set forth below.
Figure imgf000033_0001
“Me” refers to methyl modification, “Ac” to acetyl modifications, “P” to phosphorylation modifications, and “Ub” to ubiquinylations. Modifications can be mono-, di-, or tri- modifications, such as mono-, di-, or tri-methylations.
[00158] For example, the histone polypeptide can be acetylated at Lys-14 according to SEQ ID NO: 1, 2, or 3, or a fragment or variant thereof, such as to increase histone secretion and transduction.
[00159] Histone modifications can be reversibly controlled by enzymes. For example, histone acetylases are a class of enzymes that can attach an acetyl group to an amino acid on a histone protein, whereas histone deacetylases are a class of enzymes that remove acetyl groups. Embodiments described herein thus also comprise modified histone polypeptides and fragments thereof that comprise the loss of or reduced levels of one or more post-translational modification described herein. Referring to acetylation as the representative example, such a polypeptide can be referred to as hypoacetylated. The prefix “hypo-“ can refer to low levels of, or below, such as below normal.
[00160] Assays and methods to detect histone modifications are known in the art, including immunological methods and the like employing antibodies, apatamers, and immunologically active fragments of the antibodies that can bind to the histone modification of interest. Immunohistochemical and immunocytological methods can be used in detecting the modified histones or staining the cells to detect histone modifications, including the percent of cells staining for the modification. Mass spectroscopic and electrochemical means can also be used. Methods of detecting and measuring histone modifications including non-antibody- based protocols can be used, such as software programs providing for detection of epigenetic modifications.
[00161] “Immunohistochemistry” can refer to the use of antibodies or aptamers to detect proteins in biological samples such as cells and tissue sections. Methods described herein can be carried out, for example, using standard immunohistochemical techniques known in the art (reviewed in Gosling, Immunoassays: A Practical Approach, 2000, Oxford University Press). Detection can be accomplished by labeling a primary antibody or a secondary antibody with, for example, a radioactive isotope, a fluorescent label, an enzyme or any other detectable label known in the art. Visual grading of tissue sections by intensity of staining is well known in the art. Standard controls from tumor and healthy tissue samples can be used to control for variation among samples and reagents. Moreover, negative controls that do not include primary antibodies specific for the target (i.e., histone) can be used as controls.
[00162] In embodiments, the polypeptide can comprise a fragment polypeptide (i.e., “fragment”, “variant”, or “derivative”), which can refer to a short amino acid sequence of a larger polypeptide. Protein fragments can be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part of. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, about 100, about 200, and about 500 amino acids or more in length.
[00163] In embodiments, the polypeptides and fragments thereof retain at least some biological activity. Polypeptides as described herein can include fragment, variant, or derivative molecules without limitation, so long as the polypeptide still serves its function.
For example, such biological activities can comprise crossing a cell membrane, secretion from a cell, uptake by a cell, or delivery of a macromolecule. Polypeptide fragments, for example, can include proteolytic fragments, deletion fragments and fragments which more easily reach the site of action when delivered to an animal. [00164] Polypeptides and fragments thereof can comprise variant regions, including fragments as described herein, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can occur naturally, such as an allelic variant. By an “allelic variant” is intended alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Polypeptides and fragments thereof can comprise conservative or nonconservative amino acid substitutions, deletions or additions.
[00165] Polypeptides or fragments thereof can also include derivative molecules. As used herein a “derivative” of a polypeptide or a polypeptide fragment can refer to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as “derivatives” are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.
[00166] For example, the fragment can be an N-terminal fragment of histone H3, a C- terminal fragment of histone H3, or an internal fragment of histone H3 (wherein a portion of the N-terminus and a portion of the C-terminus are truncated, thus leaving a fragment corresponding to a polypeptide sequence that is internal to the full length histone H3 polypeptide). Without wishing to be bound by theory, for example, deletion of the N-terminal tail of histone H3 increases levels of extracellular H3 (i.e., histone H3 secreted from the cell). [00167] In embodiments, the fragment of histon H3 comprises a deletion of about 5 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, about 10 amino acids from SEQ
ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, about 15 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, about 20 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, about 25 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, about 50 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, about 100 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or more than about 100 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
[00168] In embodiments, the fragment polypeptide comprises a fragment of an amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identical thereto. In some embodiments, the fragment can be about 5 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 5 amino acids to about 10 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 10 amino acids to about 20 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 20 amino acids to about 30 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 30 amino acids to about 40 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 40 amino acids to about 50 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 50 amino acids to about 60 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 60 amino acids to about 70 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 70 amino acids to about 80 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 80 amino acids to about 90 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 90 amino acids to about 100 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 100 amino acids to about 110 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 110 amino acids to about 120 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 120 amino acids to about 130 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; from about 130 amino acids to about 135 amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
[00169] In embodiments, the histone polypeptide or fragment thereof exhibits at least one functional and/or biological activity. The phrase “biological activity” can refer to an in vivo activity, such as an activity within an organism or within a cell. Examples of such biological activities include, but are not limited to, crossing a cell membrane, secretion from a cell, uptake by a cell, or delivery of a macromolecule.
[00170] In embodiments, the histone polypeptide or fragment thereof can be isolated from a host cell, such as a host cell which comprises a nucleic acid encoding for and expressing the polypeptide or fragment thereof. The phrase "host cell", "host cell line," and "host cell culture" can be used interchangeably and can refer to the cells into which exogenous nucleic acid can be introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and the progeny derived therefrom without regard to the number of passages. The progeny does not need to be completely identical in nucleic acid content to a parent cell, but it can contain mutations. Mutant progeny that have the same biological function or activity as screened or selected in the originally transformed cell are included in the invention. Host cells can include any eukaryotic cell or prokaryotic cell, including but not limited to a mammalian cell, avian cell, amphibian cell, plant cell, fish cell, insect cell, bacterial cell, yeast cell, whether in vitro or in vivo.
[00171] In embodiments, the host cell can be a mammalian cell. The phrase “mammalian cell” can refer to a cell of any mammal, including humans. The phrase can refer to cells in vivo, such as, for example, in an organism or in an organ of an organism. The phrase also can refer to cells in vitro, such as, for example, cells maintained in cell culture. [00172] In embodiments, the histone polypeptide or fragment thereof can be component of a fusion protein. The phrase “fusion protein” or “fusion polypeptide” can refer to a complex formed by the fusion of a polypeptide sequence (i.e., a histone polypeptide sequence) with one or more elements. For example, the fusion protein can comprise the histone polypeptide or fragment thereof fused to at least one functional moiety. The phrase “functional moiety” can refer to an element (i.e., component) that can perform a function, for example, an imaging function, a biological function (e.g., replacing a biological function that is otherwise missing or erroneously modulated, such as due to a missing or defective protein), or a drug- delivery function. Non-limiting examples of functional moieties comprise a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof .
[00173] The phrase “therapeutic moiety” (i.e., therapeutic agent) can refer to molecule, compound, or fragment thereof that is used for the treatment of a disease or for improving the well-being of an organism or that otherwise exhibit healing power (e.g., pharmaceuticals, drugs, and the like). A therapeutic moiety can be a chemical, or fragment thereof, of natural or synthetic origin used for its specific action against disease, for example cancer. Non- limiting examples of therapeutic moieties include a therapeutic protein or polypeptide, a small molecule, or a toxin. For example, therapeutic moieties include chemotherapeutic agents, such as those described herein.
[00174] In embodiments, therapeutic moieties can replace a biological function that is otherwise missing or erroneously modulated, such as due to a missing or defective protein. For example, a therapeutic moiety can replace a missing or defective tumor suppressor protein in cancer (e.g., p53 or pVHL), or replace a missing or defective enzyme in metabolic diseases.
[00175] The term “cytotoxic moiety” can refer to molecule, compound, or fragment thereof that has a toxic or poisonous effect on cells, or that kills cells. Chemotherapy and radiotherapy are forms of cytotoxic therapy. Treating cells with a cytotoxic moiety can produce a variety of results cells can undergo necrosis, stop actively growing and dividing, or activate a genetic program of controlled cell death (i.e., apoptosis). Examples of cytotoxic moieties include, but are not limited to, SN-38, bendamustine, VDA, doxorubicin, pemetrexed, vorinostat, lenalidomide, irinotecan, ganetespib, docetaxel, 17-AAG, 5-FU, abiraterone, crizotinib, KW-2189, BUMB2, DC1, CC-1065, adozelesin or fragments ) thereof.
[00176] The term “imaging moiety” can refer to a molecule, compound, or fragment thereof that facilitates a technique and/or process used to create images or take measurements of a cell, tissue, and/or organism (or parts or functions thereof) for clinical and/or research purposes. An imaging moiety can produce, for example, a signal through emission and/or interaction with electromagnetic, nuclear, and/or mechanical (e.g., acoustic as in ultrasound) energy. An imaging moiety can be used, for example, in various radiology, nuclear medicine, endoscopy, thermography, photography, spectroscopy, and microscopy methods. In embodiments, the imaging moiety (i.e., the diagnostic agent, or imaging agent) can be radioactive, enzymatic, chemiluminescent, fluorescent, quantum dot, or others. A nonlimiting example of an imaging moiety comprises one which can be used for PET imaging, such as HSV TK, or fluorescence imaging, such as GFP.
[00177] In embodiments, the imaging moiety can comprise a “label” or “detectable moiety”. A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes ( for example, those as used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide. Labels can be conjugated directly to a biorecognition molecules, or to probes that bind these molecules, using conventional methods that are well known in the arts. Multiple labeling schemes are known in the art and permit a plurality of binding assays to be performed simultaneously. Different labels can be radioactive, enzymatic, chemiluminescent, fluorescent, quantum dot, or others. Methods of covalently or noncovalently conjugating labels to antibodies are well known to one of ordinary skill in the art. Methods of detecting proteins and modified proteins by use of labeled antibodies are also well known to persons of ordinary skill in the art.
[00178] The term “linker” can refer to a chemical moiety or bond that attaches two or more molecules. In embodiments, the two or more molecules can comprise a drug delivery moiety (i.e., a histone polypeptide or fragment thereof), a functional moiety, and/or a solid support (e.g., microparticle). Linkers can comprise contiguous chains of one or more components, such as carbon, oxygen, nitrogen, sulfur, phosphorous and combinations thereof. Linkers can connect the molecule via a covalent bond or other means, such as ionic or hydrogen bond interactions. Linkers can be cleavable, such as to release a functional moiety from the histone polypeptide once inside a target cell.
[00179] In embodiments, the linker can comprise the nucleic acid sequence:
Figure imgf000041_0001
[00180] The term “linker spacer group” can refer to atoms in the linker that provide space between the two molecules joined by the linker.
[00181] In embodiments, the linker comprises from 1 to 30 or less amino acids linked by peptide bonds. The amino acids can be selected from the 20 naturally occurring amino acids. Alternatively, non-natural amino acids can be incorporated by chemical synthesis, post- translational chemical modification or by in vivo incorporation by recombinant expression in a host cell. Some of these amino acids can be glycosylated. [00182] In embodiments amino acids of the linker are selected from glycine, alanine, proline, asparagine, glutamine, lysine, aspartate, and glutamate. In embodiments the linker can be made up of a majority of amino acids that are sterically unhindered, such as glycine, alanine and/or serine. For example, linkers can comprise polyglycine linkers, including but not limited to (Gly)3, (GlyMSEQ ID NO:5), (Gly)s (SEQ ID NO: 6). Other examples include polyalanines, poly(Gly-Ala), poly(GlymSer), poly (Glvn-Glu), poly(Glyn-Lys), poly(Glyn- Asp), and poly(Glyn-Arg) motifs. Other specific examples of linkers are (GlyjsLysiGiy )* (SEQ ID NO; 7); (GlyjsAsnGlySerlGlyjz (SEQ ID NO: 8);
(Gly)3Cy$(Gly)4 (SEQ ID NO; 9); and GlyProAsnGlyGly (SEQ ID NO: 10), Combinations of G!y and Ala are useful as are combination of Gly and Ser, Thus, in a further embodiment the peptide linker is selected from the group consisting of a glycine rich peptide, e.g. Gly-Gly- Gly: the sequences [Gly-Ser]r.(SEQ ID NO: 11), [Gly-Gly-Serjn (SEQ ID NO: 12), [Gly-Gly- Gly-Ser]n (SEQ ID NO: 13) and [Gly-Gly-Gly-Giy-Ser]n (SEQ ID NO: 14), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, [Gly-Giy-Gly Ser] i (SEQ ID NO: 15), [Gly-Gly-Gly-Gly Ser] 1 (SEQ ID NO: 16), [Gly-Gly-Gly Serb (SEQ ID NO: 17), or [Gly-Gly-Gly-Gly Ser] 3 (SEQ ID NO: 18).
[00183] In embodiments, charged linkers can be used. Such charges linkers can contain a significant number of acidic residues (e.g.. Asp, Glu, and the like), or can contain a significant number of basis residues (e.g., Lys, Arg, and the like), such that the linker has a pi lower than 7 or greater than 7, respectively. As understood by the artisan, and other things being equal, the greater the relative amount of acidic or basic residues in a given linker, the lower or higher, respectively, the pi of the linker will he. Such linkers can impart advantages to the engineered polypeptides disclosed herein, such as improving solubility and/or stability characteristics of such polypeptides at a specific pH, such as a physiological pH (e.g., between pH 7.2 and pH 7.6, inclusive), or a pH of a pharmaceutical composition comprising such polypeptides.
[00184] For example, an "acidic linker’' is a linker that has a pi of less than 7; between 6 and 7, inclusive; between 5 and 6, inclusive; between 4 and 5, inclusive, between 3 and 4, inclusive; between 2 and 3, inclusive, or between I and 2, inclusive. Similarly, a “basic linker’ is a linker that has a pi of greater than 7, between 7 and 8, inclusive; between 8 and 9, inclusive; between 9 and 10, inclusive; between 10 and i t, inclusive; between 11 and 12 inclusive, or between 12 and 13, inclusive. In certain embodiments, an acidic linker will contain a sequence that is selected from the group consisting of [Gly~Glu]a(SEQ ID NO; 19); [Gly~Gly~Glu]n (SEQ ID NO; 20): [Gly-Gly-Gly-GIu],, (SEQ ID MO; 21); [Gly-GIy-Gly-Gly- Giujn (SEQ ID NO; 22), [Gly~Asp]GSEQ ID NO: 23); [Gly-GIy-Asp]n(SEQ ID NO: 24); [Gly-Gly-Gly-Asp]n(SEQ ID NO: 24); [Giy-Gly-Gly-Gly-Asp]n(SEQ ID NO: 26) where n is I, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; for example, [Gly-Gly-Gluje (SEQ ID NO: 27). In certain embodiments, a basic linker will contain a sequence that is selected from the group consisting of [Gly-Lys];; (SEQ ID NO: 28); [Gly-Gly-Lys]«(SEQ ID NO: 29); [Giy-Gly-Gly-Lys]n (SEQ ID NO: 30); [Gly-Gly-Gly-Gly-Lysjr, (SEQ ID NO: 31), [Gly-Arg]« (SEQ ID NO: 32); [Gly- Giy-Arg]:; (SEQ ID NO: 33); [Gly-Gly-Gly-Arg]K(SEQ ID NO: 34); [Gly-Gly-Gly-Giy- Arg] n (SEQ ID NO: 35) where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; for example, [Gly-Gly- Lysjs (SEQ ID NO: 36).
[00185] Additionally, linkers can be prepared which possess certain structural motifs or characteristics, such as an a helix. For example, such a linker can contain an sequence that is selected from the group consisting of [Glu-A3a-Ala-Aia-Lys]n(SEQ ID NO: 37), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, for example, [Giu- A1 a-Ala- A! a-Ly s] 3 (SEQ ID NO: 38), [GIu-A3a~Ala-A3a~Lys]i (SEQ ID NO; 39), or [G3 u- A3 a- Ala- Ai a-Ly s ] 5 (SEQ ID NO: 40). [00186] Additionally, a non-peptidic linker can be employed to serve as the LI moiety of an engineered polypeptide described herein. For example, as understood in the art, an exemplary non-peptide linker such as a PEG linker can be so-employed. See, e g., W02000024782. In embodiments, such a PEG linker has a molecular weight ofl kDa to 1000 kDa.
[00187] It is also to be understood that linkers suitable for use in accordance with the invention can possess one or more of the characteristics and motif, such as those described herein. For example, a linker can comprise an acidic linker as well as a structural motif, such as an alpha helix. Similarly, a linker can comprise a basic linker and a structural motifi such as an alpha helix. A linker can comprise an acidic linker, a basic linker, and a structural motif, such as an a helix. Additionally, it is also to he understood that engineered polypeptides in accordance with the invention can possess more than one linker, and each such linker can possess one or more of the characteristics described herein.
[00188] The linkers described herein are exemplary', and linkers within the scope of this invention can be much longer and can include other residues.
[00189] The term “adjuvant” can refer to any substance that assists or modifies the action of a pharmaceutical, including but not limited to immune adjuvants that enhance and/or diversify the immune response to an antigen. Thus, immune adjuvants include compounds that can enhance the immune response to an antigen. An immune adjuvant can enhance body fluid and/or cellular immunity. Substances that stimulate the innate immune response are included within the definition of immune adjuvant herein.
[00190] In embodiments, the functional moiety can be a macromolecule. The term “macromolecule” can refer to peptides, proteins, and large molecules (molecular weights of 1000 Daltons or more). Non-limiting examples of macromolecules include proteins (e.g., polypeptides), fats, fatty acids or nucleic acids (e.g., oligonucleotides and polynucleotides), of biological or synthetic origin. For example, engineered polypeptides described herein have delivered cargo such as Cre recombinase (38kDa), eGFP (26.9kDa), and mCherry (28kDa). See, for example, the sequences according to SEQ ID NO: 43-SEQ ID NO: 48.
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
[00191] In embodiments, the macromolecule can be a protein or a fragment thereof. For example, the protein can be an antibody or fragment thereof, transcription factor or other protein with biological activity, a growth inhibitor (i.e., suppresses in vitro or in vivo cell growth) or a cytokine.
[00192] The term “antibody” can refer to a polypeptide from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. As a non-limiting example, the antibody or fragment thereof can be specific for an oncoprotein, such as c-Myc. [00193] The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains can be classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes,
IgG, IgM, IgA, IgD and IgE, respectively. The antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
[00194] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The teens variable light chain (VL) and variable heavy chain (VH) can refer to these light and heavy chains respectively.
[00195] Antibodies exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments, such as produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 can be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments can be synthesized de novo, such as chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments, such as those produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)). For example, a single chain antibody can be fused to a histone polypeptide, such as histone H3.
[00196] The term “cytokine” can refer to a molecule that mediates and/or regulates a biological or cellular function or process (e.g. immunity, inflammation, and hematopoiesis). The term “cytokine” includes “lymphokines,” “chemokines,” “monokines,” and “interleukins”. Examples of useful cytokines include, but are not limited to, GM-CSF, IL-la, IL-10, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IFN-a, IFN-b, IFN-g, MIP-la, MIR-Ib, TGF-b, TNF-a, and TNF-b. The term “cytokine” as used herein is meant to also include cytokine variants comprising one or more amino acid mutations in the amino acid sequences of the corresponding wild-type cytokine, such as for example the IL-2 variants described in Sauve et al., Proc Natl Acad Sci USA 88, 4636-40 (1991); Hu et al., Blood 101, 4853-4861 (2003) and US Pat. Publ. No. 2003/0124678; Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000); Heaton et al., Cancer Res 53, 2597-602 (1993) and U.S. Pat. No. 5,229,109; US Pat. Publ. No. 2007/0036752; WO 2008/0034473; WO 2009/061853; or in WO 2012/107417.
[00197] Aspects of the invention are also drawn to nucleic acids encoding a histone polypeptide or fragment thereof. The phrase “nucleic acid” can refer to deoxyribonucleotides or ribonucleotides and polymers thereof, such as those in single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base, such as a purine base or pyrimidine base. The term also encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. In embodiments, a nucleic acid sequence can also encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the reference sequence explicitly indicated.
[00198] For example, the human histone H3 nucleic acid sequence comprises
Figure imgf000051_0001
BOLDED “ATG” comprises start codon.
[00199] The term “oligonucleotide” can refer to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, for example more than three, and, in embodiments, more than ten. There is no precise upper limit on the size of an oligonucleotide. In embodiments, an oligonucleotide can be shorter than about 250 nucleotides, shorter than about 200 nucleotides, or shorter than about 100 nucleotides. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide can be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. [00200] In embodiments, the nucleic acid comprises a nucleotide sequence according to 4 or a fragment thereof, or a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identical thereto.
[00201] In embodiments, the nucleic acid comprises a “nucleic acid fragment”, which can refer to a portion of a larger nucleic acid molecule.
[00202] Aspects of the invention are further drawn to a cell comprising the nucleic acids described herein. As described herein, the cell can be referred to as a host cell. For example, the phrase "host cell", "host cell line," and "host cell culture" can be used interchangeably and can refer to the cells into which exogenous nucleic acid can be introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and the progeny derived therefrom without regard to the number of passages. The progeny does not need to be completely identical in nucleic acid content to a parent cell, but it can contain mutations. Mutant progeny that have the same biological function or activity as screened or selected in the originally transformed cell are included in the invention. Host cells can include any eukaryotic cell or prokaryotic cell, including but not limited to a mammalian cell, avian cell, amphibian cell, plant cell, fish cell, insect cell, bacterial cell, yeast cell, whether in vitro or in vivo.
[00203] In embodiments, the host cell can be a mammalian cell. The phrase “mammalian cell” can refer to a cell of any mammal, including humans. The phrase can refer to cells in vivo, such as, for example, in an organism or in an organ of an organism. The phrase also can refer to cells in vitro, such as, for example, cells maintained in cell culture.
[00204] The engineered polypeptides described herein can be designed at the amino acid level. These sequences can then be back translated using a variety of software products known in the art such that the nucleotide sequence is optimized for expression, such as in a given host (for example, based protein expression, codon optimization, restriction site content). For example, the nucleotide sequence can be optimized for E. coli based protein expression and for restriction site content. Based on the nucleotide sequence of interest, overlapping oligonucleotides can be provided for multistep PCR, as known in the art. These oligonucleotides can be used in multiple PCR reactions under conditions well known in the art to build the cDNA encoding the protein of interest. For one example is 1 x Amplitaq Buffer, 1.3 mM MgC12, 200 uM dNTPs, 4 U Amplitaq Gold, 0.2 uM of each primer (AmpliTaq Gold, ABI), with cycling parameters: (94 C:30 s, 58 C:1 min, 72 C: 1 min), 35 cycles. The skilled artisan will recognize that there are multiple ways to generate genes of interest, in addition to synthesizing an entire cDNA of interest.
[00205] Restriction sites can be added to the ends of the PCR products for use in vector ligation as known in the art. Specific sites can include Ndel and Xhol, such that the cDNA can then be in the proper reading frame in a pET45b expression vector (Novagen). By using these sites, any N-terminal His Tag that are in this vector can be removed as the translation start site can then be downstream of the tag. Once expression constructs are completed, verification can be conduct by sequencing using e.g., T7 promoter primer, T7 terminator primer and standard ABI BigDye Term v3.1 protocols as known in the art. Sequence information can be obtained from e.g., an ABI 3730 DNA Analyzer and can be analyzed using Vector NTI v.10 software (Invitrogen). Expression constructs can be designed in a modular manner such that linker sequences can be easily cut out and changed, as known in the art. The skilled artisan will recognize that expression constructs can also be designed without the use of restriction enzymes, such as using homology based methods like In-Fusion Cloning and Gibson Assembly.
[00206] Protease recognition sites, known in the art, can be incorporated into constructs useful for the design, construction, manipulation and production of recombinant engineering polypeptides described herein. [00207] The engineered polypeptides described herein can be prepared using biological, chemical, and/or recombinant DNA techniques that are known in the art. Exemplary methods are described herein and in U.S. Pat. No. 6,872,700; WO 2007/139941; WO 2007/140284; WO 2008/082274; WO 2009/011544; and US Publication No. 2007/0238669, the disclosures of which are incorporated herein by reference in their entireties. Other methods for preparing the compounds are set forth herein.
[00208] The engineered polypeptides described herein can be prepared using standard solid-phase peptide synthesis techniques, such as an automated or semiautomated peptide synthesizer. For example, using such techniques, an alpha-N-carbamoyl protected amino acid and an amino acid attached to the growing peptide chain on a resin are coupled at RT in an inert solvent (e.g., dimethylformamide, N-methylpyrrolidinone, methylene chloride, and the like) in the presence of coupling agents (e.g., dicyclohexylcarbodiimide, 1-hydroxybenzo- triazole, and the like) in the presence of a base (e.g., diisopropylethylamine, and the like).
The alpha-N-carbamoyl protecting group is removed from the resulting peptide-resin using a reagent (e.g., trifluoroacetic acid, piperidine, and the like) and the coupling reaction repeated with the next N-protected amino acid to be added to the peptide chain. Suitable N-protecting groups are well known in the art, such as t-butyloxy carbonyl (tBoc) fluorenylmethoxycarbonyl (Fmoc), and the like. The solvents, amino acid derivatives and 4- methylbenzhydryl-amine resin used in the peptide synthesizer can be purchased from Applied Biosystems Inc. (Foster City, Calif.).
[00209] For chemical synthesis solid phase peptide synthesis can be used for the engineered polypeptides, since in general solid phase synthesis is a straightforward approach with excellent scalability to commercial scale, and can be compatible with relatively long engineered polypeptides. Solid phase peptide synthesis can be carried out with an automatic peptide synthesizer (Model 430A, Applied Biosystems Inc., Foster City, Calif.) using the NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry (See Applied Biosystems User's Manual for the ABI 430A Peptide Synthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49- 70, Applied Biosystems, Inc., Foster City, Calif.) with capping. Boc-peptide-resins can be cleaved with HF (-5° C. to 0° C., 1 hour). The peptide can be extracted from the resin with alternating water and acetic acid, and the filtrates lyophilized. The Fmoc-peptide resins can be cleaved according to standard methods (e.g., Introduction to Cleavage Techniques,
Applied Biosystems, Inc., 1990, pp. 6-12). Peptides can also be assembled using an Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.).
[00210] The compounds described herein can also be prepared using recombinant DNA techniques using methods known in the art, such as Sambrook et ak, 1989, MOLECULAR CLONING: A LABORATORY MANUAL, 2d Ed., Cold Spring Harbor. Non-peptide compounds can be prepared by art-known methods. For example, phosphate-containing amino acids and peptides containing such amino acids, can be prepared using methods known in the art, such as described in Bartlett et al, 1986, Biorg. Chem. 14:356-377.
[00211] The engineered polypeptides can alternatively be produced by recombinant techniques well known in the art. See, e.g., Sambrook et ak, 1989 (Id.). These engineered polypeptides produced by recombinant technologies can be expressed from a polynucleotide. One skilled in the art will appreciate that the polynucleotides, including DNA and RNA, that encode such engineered polypeptides can be obtained from the wild-type cDNA, taking into consideration the degeneracy of codon usage, and can be further engineered to incorporate the indicated substitutions. These polynucleotide sequences can incorporate codons facilitating transcription and translation of mRNA in microbial hosts. Such manufacturing sequences can readily be constructed according to the methods well known in the art. See, e.g., WO 83/04053, incorporated herein by reference in its entirety.. The polynucleotides described herein can also optionally encode an N-terminal methionyl residue. Non-peptide compounds useful in the invention can be prepared by art-known methods. For example, phosphate-containing amino acids and peptides containing such amino acids can be prepared using methods known in the art. See, e.g., Bartlett and Landen, 1986, Bioorg. Chem. 14: 356- 77.
[00212] A variety of expression vector/host systems can be utilized to contain and express an engineered polypeptide coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems. Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), WI 38, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols for the recombinant expression of the protein are described herein and/or are known in the art.
[00213] As such, polynucleotide sequences are useful in generating new and useful viral and plasmid DNA vectors, new and useful transformed and transfected prokaryotic and eukaryotic host cells (including bacterial, yeast, and mammalian cells grown in culture), and new and useful methods for cultured growth of such host cells that can express the engineered polypeptides. The polynucleotide sequences encoding engineered polypeptides herein can be useful for gene therapy in instances where underproduction of engineered polypeptides can be alleviated, or the need for increased levels of such can be met.
[00214] This invention also provides for processes for recombinant DNA production of the engineered polypeptides. Provided is a process for producing the engineered polypeptides from a host cell containing nucleic acids encoding the engineered polypeptide comprising: (a) culturing the host cell containing polynucleotides encoding the engineered polypeptide under conditions facilitating the expression of the DNA molecule; and (b) obtaining the engineered polypeptide.
[00215] Host cells can be prokaryotic or eukaryotic and include bacteria, mammalian cells (such as Chinese Hamster Ovary (CHO) cells, monkey cells, baby hamster kidney cells, cancer cells or other cells), yeast cells, and insect cells.
[00216] Mammalian host systems for the expression of the recombinant protein also are well known to those of skill in the art. Host cell strains can be chosen for their ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cleaves a “prepro” form of the protein, can also be important for correct insertion, folding and/or function. Different host cells, such as CHO, HeLa, MDCK, 293, W138, and the like, have specific cellular machinery and characteristic mechanisms for such post-translational activities, and can be chosen to ensure the correct modification and processing of the introduced foreign protein.
[00217] Alternatively, a yeast system can be employed to generate the engineered polypeptides of the invention. The coding region of the engineered polypeptides DNA is amplified by PCR. A DNA encoding the yeast pre-pro-alpha leader sequence is amplified from yeast genomic DNA in a PCR reaction using one primer containing nucleotides 1-20 of the alpha mating factor gene and another primer complementary to nucleotides 255-235 of this gene (Kurjan and Herskowitz, 1982, Cell, 30:933-43). The pre-pro-alpha leader coding sequence and engineered polypeptide coding sequence fragments are ligated into a plasmid containing the yeast alcohol dehydrogenase (ADH2) promoter, such that the promoter directs expression of a fusion protein consisting of the pre-pro-alpha factor fused to the mature engineered polypeptide. As taught by Rose and Broach, Meth. Enz. 185: 234-79, Goeddel ed., Academic Press, Inc., San Diego, Calif. (1990), the vector further includes an ADH2 transcription terminator downstream of the cloning site, the yeast “2-micron” replication origin, the yeast leu-2d gene, the yeast REP1 and REP2 genes, the E. coli beta-lactamase gene, and an E. coli origin of replication. The beta-lactamase and leu-2d genes provide for selection in bacteria and yeast, respectively. The leu-2d gene also facilitates increased copy number of the plasmid in yeast to induce higher levels of expression. The REPl and REP2 genes encode proteins involved in regulation of the plasmid copy number.
[00218] The DNA construct described in the preceding paragraph is transformed into yeast cells using a known method, e.g., lithium acetate treatment (Steams et al., 1990, Meth. Enz. 185: 280-297). The ADH2 promoter is induced upon exhaustion of glucose in the growth media (Price et al., 1987, Gene 55:287). The pre-pro-alpha sequence effects secretion of the fusion protein from the cells. Concomitantly, the yeast KEX2 protein cleaves the pre-pro sequence from the mature engineered polypeptides (Bitter et al., 1984, Proc. Natl. Acad. Sci. USA 81:5330-5334).
[00219] Engineered polypeptides of the invention can also be recombinantly expressed in yeast, e.g., Pichia, using a commercially available expression system, e.g., the Pichia Expression System (Invitrogen, San Diego, Calif.), following the manufacturer's instructions. This system also relies on the pre-pro-alpha sequence to direct secretion, but transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction by methanol. The secreted engineered polypeptide is purified from the yeast growth medium by, e.g., the methods used to purify said engineered polypeptide from bacterial and mammalian cell supernatants. [00220] Alternatively, the DNA encoding an engineered polypeptide can be cloned into a baculovirus expression vector, e.g. pVL1393 (PharMingen, San Diego, Calif.). This engineered-polypeptide-encoding vector is then used according to the manufacturer's directions (PharMingen) or known techniques to infect Spodoptera frugiperda cells, grown for example in sF9 protein-free media, and to produce recombinant protein. The protein is purified and concentrated from the media using methods known in the art, e.g. a heparin- Sepharose column (Pharmacia, Piscataway, N.J.) and sequential molecular sizing columns (Amicon, Beverly, Mass.), and resuspended in appropriate solution, e.g. PBS. SDS-PAGE analysis can be used to characterize the protein, for example by showing a single band that confirms the size of the engineered polypeptide, as can full amino acid amino acid sequence analysis, e.g. Edman sequencing on a Proton 2090 Peptide Sequencer, or confirmation of its N-terminal sequence.
[00221] For example, the DNA sequence encoding the predicted mature engineered polypeptide can be cloned into a plasmid containing a promoter and, optionally, a leader sequence (see, e.g., Better et ak, 1988, Science 240:1041-1043). The sequence of this construct can be confirmed by automated sequencing. The plasmid can then be transformed into E. coli, strain MCI 061, using standard procedures employing CaC12 incubation and heat shock treatment of the bacteria (Sambrook et ak, Id.). The transformed bacteria are grown in LB medium supplemented with carbenicillin, and production of the expressed protein is induced by growth in a suitable medium. The leader sequence can affect secretion of the mature engineered polypeptide and be cleaved during secretion. The secreted recombinant engineered polypeptide can be purified from the bacterial culture media by the method described herein.
[00222] In another embodiment, the engineered polypeptides can be expressed in an insect system. Insect systems for protein expression are well known to those of skill in the art. In one such system, Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The engineered polypeptide coding sequence is cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of an engineered polypeptide will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which engineered polypeptide of the invention is expressed (Smith et al., 1983, J. Virol. 46:584; Engelhard et al., 1994, Proc. Natl. Acad. Sci. USA 91:3224-3227).
[00223] In another example, the DNA sequence encoding the engineered polypeptides can be amplified by PCR and cloned into an appropriate vector, for example, pGEX-3X (Pharmacia, Piscataway, N. J.). The pGEX vector is designed to produce a fusion protein comprising glutathione-S-transferase (GST), encoded by the vector, and a protein encoded by a DNA fragment inserted into the vector's cloning site. The primers for the PCR can be generated to include, for example, an appropriate cleavage site. The recombinant fusion protein can then be cleaved from the GST portion of the fusion protein. The pGEX- 3X/engineered polypeptide construct is transformed into E. coli XL-1 Blue cells (Stratagene, La Jolla, Calif.), and individual transformants are isolated and grown at 37° C. in LB medium (supplemented with carbenicillin) to an optical density at wavelength 600 nm of 0.4, followed by further incubation for 4 hours in the presence of 0.5 mM Isopropyl beta-D- thiogalactopyranoside (Sigma Chemical Co., St. Louis, Mo.). Plasmid DNA from individual transformants is purified and partially sequenced using an automated sequencer to confirm the presence of the engineered polypeptide-encoding gene insert in the proper orientation. [00224] The fusion protein, when produced as an insoluble inclusion body in the bacteria, can be purified as described herein or as follows. Cells are harvested by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/mL lysozyme (Sigma Chemical Co.) for 15 min. at RT. The lysate is cleared by sonication, and cell debris is pelleted by centrifugation for 10 min. at 12,000xg. The fusion protein- containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, layered over 50% glycerol, and centrifuged for 30 min. at 6000xg. The pellet is resuspended in standard phosphate buffered saline solution (PBS) free of Mg++ and Ca++. The fusion protein is further purified by fractionating the resuspended pellet in a denaturing SDS polyacrylamide gel (Sambrook et al., supra). The gel is soaked in 0.4 M KC1 to visualize the protein, which is excised and electroeluted in gel-running buffer lacking SDS. If the GST/engineered polypeptide fusion protein is produced in bacteria as a soluble protein, it can be purified using the GST Purification Module (Pharmacia Biotech).
[00225] The fusion protein can be subjected to digestion to cleave the GST from the mature engineered polypeptide. The digestion reaction (20-40 pg fusion protein, 20-30 units human thrombin (4000 U/mg (Sigma) in 0.5 mL PBS) is incubated 16-48 hrs. at RT and loaded on a denaturing SDS-PAGE gel to fractionate the reaction products. The gel is soaked in 0.4 M KC1 to visualize the protein bands. The identity of the protein band corresponding to the molecular weight of the engineered polypeptide can be confirmed by partial amino acid sequence analysis using an automated sequencer (Applied Biosystems Model 473A, Foster City, Calif.).
[00226] In embodiments of recombinant expression of the engineered polypeptides of the invention, 293 cells can be co-transfected with plasmids containing the engineered polypeptides cDNA in the pCMV vector (5' CMV promoter, 3' HGH poly A sequence) and pSV2neo (containing the neo resistance gene) by the calcium phosphate method. In one embodiment, the vectors can be linearized with Seal prior to transfection. Similarly, an alternative construct using a similar pCMV vector with the neo gene incorporated can be used. Stable cell lines are selected from single cell clones by limiting dilution in growth media containing 0.5 mg/mL G418 (neomycin-like antibiotic) for 10-14 days. Cell lines are screened for engineered polypeptides expression by ELISA or Western blot, and high- expressing cell lines are expanded for large scale growth.
[00227] In embodiments, viral delivery systems can be used to transduce cells. For example, lentiviral infection (i.e., lentiviral transduction) can be used to introduce an exogenous polynucleotide into the genome of a cell. The use of lentiviral vectors permits stable expression of the polynucleotide of interest. For example, lentiviral transduction can be performed by incubating cells to be transduced with a lentiviral vector carrying at least one polynucleotide encoding a polypeptide of interest.
[00228] In embodiments, the transformed cells are used for long-term, high-yield protein production and as such stable expression is desirable. Once such cells are transformed with vectors that contain selectable markers along with the expression cassette, the cells can be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The selectable marker can be designed to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell.
[00229] A number of selection systems can be used to recover the cells that have been transformed for recombinant protein production. Such selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside, G418; also, that confers resistance to chlorsulfuron; hygro, that confers resistance to hygromycin; or PAC, that confers resistance to puromycin. Additional selectable genes that can be useful include trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine. Markers that give a visual indication for identification of transformants include anthocyanins, beta- glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin.
[00230] The engineered polypeptides of the invention can be produced using a combination of both automated peptide synthesis and recombinant techniques. Engineered polypeptides can be made synthetically or recombinantly and then, in embodiments, ligated together using methods known in the art, such as “native chemical ligation” and known variations thereof in which an amide bond is formed joining the parent compounds. See for example U.S. Pat. No. 6,326,468. In other embodiments, for example, an engineered polypeptide of the invention can contain a combination of modifications including deletion, substitution, insertion and derivatization by PEGylation (or other moiety, e.g. polymer, fatty acyl chain, C-terminal amidation). Such an engineered polypeptide can be produced in stages. In the first stage, an intermediate engineered polypeptide containing the modifications of deletion, substitution, insertion, and any combination thereof, can be produced by recombinant techniques as described. Then after an optional purification step as described herein, the intermediate engineered polypeptide is PEGylated (or subjected to other chemical derivatization, e.g., acylation, C-terminal amidation) through chemical modification with an appropriate PEGylating reagent (e.g., fromNeKtar Transforming Therapeutics, San Carlos, Calif.) to yield the engineered polypeptide derivative. One skilled in the art will appreciate that the procedure described herein can apply to an engineered polypeptide containing a combination of modifications selected from deletion, substitution, insertion, derivation, and other means of modification well known in the art. [00231] C-terminal amidation can be achieved by use of a glycine amino acid-C-terminally extended precursor, synthesized for example in yeast (e.g. Pichia) as alpha-factor fusion protein that will be secreted into culture medium. After purification, the C-terminal glycine of the engineered polypeptide precursor can be converted to amide by enzymatic amidation, e.g. peptidylglycine alpha-amidating monooxygenase (PAM). See e.g., Cooper et al., 1989, Biochem. Biophys. Acta, 1014:247-258. See also U.S. Pat. No. 6,319,685, which teaches methods for enzymatic amidation, including an alpha-amidating enzyme from rat being sufficiently pure in alpha-amidating enzyme to exhibit a specific activity of at least about 25 mU per mg of protein, and being sufficiently free of proteolytic impurities to be suitable for use with substrates purified from natural sources or produced by recombinant DNA techniques.
[00232] Peptides can be purified by any number of methods known in the art, including as described herein. In one method peptides are purified by RP-HPLC (preparative and analytical) using a Waters Delta Prep 3000 system. A C4, C8 or Cl 8 preparative column (IOm, 2.2x25 cm; Vydac, Hesperia, Calif.) can be used to isolate peptides, and purity can be determined using a C4, C8 or C18 analytical column (5m, 0.46x25 cm; Vydac). Solvents (A=0.1% TF A/water and B=0.1% TFA/CH3CN) can be delivered to the analytical column at a flow rate of 1.0 ml/min and to the preparative column at 15 ml/min. Amino acid analyses can be performed on the Waters Pico Tag system and processed using the Maxima program. Peptides can be hydrolyzed by vapor-phase acid hydrolysis (115° C., 20-24 h). Hydrolysates can be derivatized and analyzed by standard methods (Cohen et al, THE PICO TAG METHOD: A MANUAL OF ADVANCED TECHNIQUES FOR AMINO ACID ANALYSIS, pp. 11-52, Millipore Corporation, Milford, Mass. (1989)). Fast atom bombardment analysis can be carried out by M-Scan, Incorporated (West Chester, Pa.). Mass calibration can be performed using cesium iodide or cesium iodide/glycerol. Plasma desorption ionization analysis using time of flight detection can be carried out on an Applied Biosystems Bio-Ion 20 mass spectrometer.
[00233] Methods are available for assaying the level of protein expression by a host cell. Procedures useful for assaying the level of protein expression by a host cell are exemplified in the following typical protocol. About 25 pi BL21 E. coli cells are transformed with 2 ul plasmid DNA (expression vector for the engineered polynucleotide). Cells can be plated and incubated overnight at 37 degrees C. or at room temperature (RT) over a 48-hr period. A single colony can be selected and used to grow starter culture in 4 ml LB media with appropriate antibiotic for '6 hrs. Glycerol stocks can be prepared by adding 100 ul 80% sterile glycerol to 900 ul stock, which can then be mixed gently and stored at -80 C. A 250 mΐ sample can be removed for TCP uninduced sample. An aliquot, for example, 2 ml of Magic media containing appropriate antibiotic can be inoculated with 5 mΐ starter culture, which can then be incubated overnight (up to 24 hrs) at 37 C, 300 rpm. As known in the art, Magic Media is autoinducing. Alternatively, 60 ml Magic Media containing appropriate antibiotic can be inoculated with 60 mΐ starter culture in a 250 ml or 125 ml Thompson flask, which can then be incubated overnight (up to 24 hrs) at 30 C, 300 rpm. After incubation, 250 pL culture can be removed from each tube and the cells pelleted. The cell can be resuspended in 1 ml 50 mM Tris pH 8, 150 mM NaCl, to which can be added 0.1 volumes (100 ul) POP culture reagent and 1 mΐ r-lysozyme (1:750 dilution in r-lysozyme buffer). The mixture can be mixed well and incubated at least 10 min at RT. The preparation can then be centrifuge 10 min at 14000xG. The supernatant (soluble fraction) can be removed and retained, and samples can be prepared for gel analysis (15 m1+5 mΐ LDS). The remaining inclusion body pellet can be resuspended in 1 ml 1% SDS with sonication. The sample can be prepared for gel analysis (15 ul+5 mΐ LDS). For uninduced samples, 1.0 volumes POP culture reagent and 1 mΐ r- lysozyme (1:750 dilution in r-lysozyme buffer) can be added. The mixture can be mixed well and incubated at least 10 min at RT. In embodiments, these samples do not need to be centrifuged. The sample can then be prepared for gel analysis (15 m1+5 mΐ LDS). NU-PAGE gels (4-12%) non-reduced in 1 xMES buffer can be run and stained with Simply Blue microwave protocol. Destaining can be conducted overnight, as known in the art. A gel image can be retained, and analyzed to determine protein expression levels.
[00234] For engineered polypeptides that are found in the inclusion body fraction, the following procedure can be beneficial. The cell pellet can be resuspended in a minimum of 100 ml Lysis buffer for each 50 ml culture. Upon the addition of 30 ml, a 10 ml pipette can be used to resuspend, then the tube can be washed out with an additional 70 ml. The resuspended cell solution can be multiply run, e.g., 4 passes, through a microfluidizer at 100 PSI (min) taking care to keep chamber in ice water through the entire process. The fluidized slurry can be centrifuged at 14000xg, 20 min (e.g., JLA 10.5, 10,000 rpm). The inclusion body pellet can be resuspended on ice in chilled lysis buffer with stir bar and stir plate for 1 hour at 4°C after disruption with pipette tip. The pellet can be resuspended a second time in distilled EhO with stir bar and stir plate for 1 hour at 4°C after disruption with pipette tip, followed by centrifugation at 14000xg, 15 min. The supernatant can be removed and discarded. The resultant can be stored at -80°C.
[00235] As described herein, numerous methods are known for isolation of expressed polypeptides. The following is one example. Inclusion body pellets can be solubilized in appropriate volume of solubilization buffer (8M urea or 8M guanidine, 50 mM Tris, 10 mM DTT, pH 7.75) for 1 hour at RT. The solubilized pellets can be centrifuged for 20 min at 27 000 g. Filtered (e.g., 0.4 um) supernatant can be transferred drop by drop into appropriate volume of refolding buffer (50 mM Tris-HCl, 1 M urea, 0.8 M arginine, 4 mM cysteine, 1 mM cystamine; pH 8) at RT. The result can then be placed at 4° C. overnight or longer with gentle mixing. Samples can be concentrated and run on a gel filtration column (Superdex™ 75 26/60) at 1-2 ml/min in 4 C environment using a GE Healthsciences AKTAFPLC™. Appropriate protein containing fractions can be identified via SDS-PAGE, pooled and run through a second gel filtration column. Pooled protein can then be concentrated in Amicon filter to appropriate concentration and assayed for endotoxin levels using, e.g., Endosafe® PTS Reader (Charles River), as known in the art. Once a protein sample has passed the endotoxin criteria, it can be sterile filtered, dispensed into aliquots and run through quality control assays. Quality control assays can include analytical HPLC-SEC, non-reducing SDS PAGE and RP HPLC-MS to obtain approximate mass. Proteins can be obtained in 1 xPBS (137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM disodium phosphate, 1.4 mM monopotassium phosphate, pH7.2), distributed into aliquots and flash frozen for storage at -70 to -80° C.
[00236] Numerous methods are also known for isolation of expressed polypeptides from conditioned media. For example, a host cell (e.g., a mammalian host cell) can be engineered to express and secrete an engineered histone polypeptide described herein. The histone polypeptide can then be purified from the conditioned media using conventional techniques, such as immunoprecipitation, affinity chromatography, polypeptide A-sepharose, gel electrophoresis, and the like.
[00237] Pharmaceutical Composition
[00238] Aspects of the invention are also drawn to pharmaceutical compositions. For example, the pharmaceutical composition can comprise a therapeutic or diagnostic component and a pharmaceutically acceptable carrier, accipient or diluent.
[00239] In embodiments, pharmaceutical compositions can comprise histone polypeptides or fragments thereof, or nucleic acids encoding the same, in combination with a pharmaceutically acceptable excipient (e.g., carrier). The term “pharmaceutically acceptable carrier” can refer to pharmaceutical excipients, for example, pharmaceutically, physiologically, acceptable organic or inorganic carrier substances suitable for administration to a subject that do not deleteriously react with the active agent. Suitable pharmaceutically acceptable carriers include water, salt solutions (e.g., Ringer's solution and the like), alcohols, oils, gelatins, and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, and polyvinyl pyrrolidine. Such preparations can be sterilized and, in embodiments, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
[00240] The phrase “therapeutic agent” or “therapeutic component” can refer to an agent or component that can induce a biological effect in vivo and/or in vitro. The biological effect can be useful for treating and/or preventing a condition, disorder, or disease in a subject or patient.
[00241] The phrase “diagnostic component can refer to a portion of a molecule that allows for the detection, imaging, and/or monitoring of the presence and/or progression of a condition, pathological disorder, and/or disease.
[00242] In embodiments, the therapeutic and/or diagnostic component can be an isolated or recombinant histone polypeptide or fragment thereof as described herein, a fusion protein as described herein, a cell as described herein, and/or a nucleic acid as described herein.
[00243] In embodiments, the pharmaceutical composition can further comprise at least one additional active agent. The phrase “additional active agent” can refer to an agent useful alone, administered simultaneously, administered sequentially, or in combination with one or more additional agents, in the treatment, prophylaxis or palliative care of a subject afflicted with a disease or disorder. [00244] In embodiments, the additional active agent can be employed in the compositions in an amount previously employed alone as a“standard of care”. In embodiments, the additional active agent can be employed in the compositions in less than an amount previously employed alone as a“standard of care”. In embodiments, the additional active agent can be co-administered or employed in a composition of the disclosure in an amount effective to cause measurable reduction of a symptom or sign of a disease, disorder or condition.
[00245] For example, the additional active agent can include conventional cancer therapeutics such as chemotherapeutic agents, cytokines, chemokines, and radiation therapy. The majority of chemotherapeutic drugs can he divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. These drugs can affect cell division or DMA synthesis and function in some way. Additional therapeutics include monoclonal antibodies and the new tyrosme kinase inhibitors e.g. imatmib mesylate, which directly targets a molecular abnormality in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors).
[00246] Representative chemotherapeutic agents include, but are not limited to cisplatin, carbopiatm, oxahpiatin, mechiorethamine, cyclophosphamide, chlorambucil, vincristine, vinblastine, vinorelbme, vindesine, taxol and derivatives thereof, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, epipodophyll otoxins, trastuzumab, ceiuximab, and ntuximab, bevacizumab, and combinations thereof.
[00247]
[00248] Drus-Delivery Platform
[00249] Aspects of the invention are also drawn to drug delivery platforms comprising an isolated or recombinant histone polypeptide or fragment as described herein. [00250] The phrase “drug delivery platform” can refer to a system to facilitate the prevention and/or treatment of a disease by functioning as the primary structural component (can be referred to as the “backbone”, “scaffold”, or “carrier”) for a functional moiety (such as an imaging moiety or a functional moiety). For example, the functional component can be loaded, infused, formed, fused to, conjugated to, or otherwise incorporated into the structural component (e.g., the histone polypeptide or fragment thereof).
[00251] For example, the drug-deliver platform can comprise a fusion protein comprising a histone polypeptide or fragment thereof and at least one functional moiety. As described herein, the phrase “functional moiety” can refer to an element (i.e., component) that can perform a function, for example, an imaging function or a drug-delivery function. Non- limiting examples of functional moieties comprise a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof .
[00252]
[00253] Therapeutic and Diagnostic Delivery Methods
[00254] Aspects of the invention are also drawn towards methods for delivery of a therapeutic and/or diagnostic molecule to a cell. The phrase “delivery to a cell” or “cell delivery” can refer to introduction of therapeutic and/or diagnostic molecule as described herein (e.g., a polypeptide, fusion protein, or nucleic acid) into a cell, such as for therapeutic purposes.
[00255] Further, aspects of the invention are drawn towards methods of transducing a cell with a therapeutic and/or diagnostic molecule. The phrase “transducing a cell” or “cell transduction” can refer to the process of introducing a therapeutic and/or diagnostic molecule as described herein (e.g., a polypeptide, fusion protein, or nucleic acid) into a cell, such as for therapeutic purposes [00256] For example, embodiments comprise contacting a cell with an isolated or recombinant histone polypeptide or fragment thereof as described herein, wherein the polypeptide or fragment thereof is fused to a therapeutic and/or diagnostic component (e.g., cargo). Such methods can be used to deliver therapeutic and/or diagnostic molecules to a cell. In embodiments, the cell can be a cancer cell or the cell can be a non-cancer cell. In embodiments, the cell can be a suspected of being a cancer cell.
[00257] The phrase “cancer cell” can refer to a cell undergoing early, intermediate or advanced stages of multi-step neoplastic progression, such as that previously described (Pitot et al., Fundamentals of Oncology, 15-28 (1978)). This includes cells in early, intermediate and advanced stages of neoplastic progression including “pre-neoplastic cells (i.e., “hyperplastic cells and dysplastic cells), and neoplastic cells in advanced stages of neoplastic progression of a dysplastic cell.
[00258] “Cancer” includes cells that are not metastatic or are metastatic. The major types of cancer are carcinomas, sarcomas, melanomas, lymphoma, and leukemias. Carcinomas originate in the skin, lungs, breasts, pancreas, and other organs and glands. Lymphomas are cancers of lymphocytes. Leukemia is cancer of the blood. It does not usually form solid tumors. Sarcomas arise in bone, muscle, fat, blood vessels, cartilage, or other soft or connective tissues of the body. Melanomas are cancers that arise in the cells that make the pigment in skin. Non-limiting examples of cancers include ovarian cancer, breast cancer, lung cancer, prostate cancer, cervical cancer, pancreatic cancer, colon cancer, stomach cancer, esophagus cancer, mouth cancer, tongue cancer, gum cancer, skin cancer (e.g., melanoma, basal cell carcinoma, Kaposi's sarcoma, etc.), muscle cancer, heart cancer, liver cancer, bronchial cancer, cartilage cancer, bone cancer, testis cancer, kidney cancer, endometrium cancer, uterus cancer, bladder cancer, bone marrow cancer, lymphoma cancer, spleen cancer, thymus cancer, thyroid cancer, brain cancer, neuron cancer, mesothelioma, gall bladder cancer, ocular cancer (e.g., cancer of the cornea, cancer of uvea, cancer of the choroids, cancer of the macula, vitreous humor cancer, etc.), joint cancer (such as synovium cancer), glioblastoma, lymphoma, and leukemia. In an embodiment, the cancer comprises one or more of a colon cancer, colorectal cancer, gastrointestinal cancer, breast cancer, bladder cancer, kidney cancer, leukemia, brain cancer, sarcoma, astrocytoma, acute myelogenous leukemia (AML), and diffuse large B-lymphoma. [00259] The phrase “cell suspected of being a cancer cell” can refer to a cell which has not yet been identified to be a cancer cell but for which there is some clinical reason to suspect the cell can be a cancer cell.
[00260]
[00261] Methods of Treatment
[00262] Aspects of the invention are further drawn towards methods of treating a subject afflicted with a disease.
[00263] The term “treat” or “treatment” can refer to the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. [00264] The term “prevent,” “preventing,” or “prevention” does not require absolute forestalling of the condition or disease but can also include a reduction in the onset or severity of the disease or condition.
[00265] The terms “subject” or “individual” or “animal” or “patient” or “mammal,” can refer to any subject for whom diagnosis, prognosis, or therapy is needed, as described herein. Mammalian subjects can include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In certain embodiments, the mammal is a human subject.
[00266] The term “disease” or “condition” can refer to a state of being or health status of a patient or subject that can be treated with the compounds or methods provided herein. In embodiments, the disease or condition comprises cancer. The term “cancer” can refer to cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including but not limited to solid tumors and lymphoid cancers, kidney, breast, lung, kidney, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, esophagus, and liver cancer, lymphoma, including but not limited to non-Hodgkins and Hodgkins lymphoma, leukemia, and multiple myeloma.
[00267] Other types of cancers, which can include malignant primary or secondary tumors, include, but are not limited to, bone cancer, cancer of the larynx, gall bladder, rectum, head and neck, bronchi, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteosarcoma, Ewin's sarcoma, reticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hair-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuromas, cervical cancer, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, rhabdomyosarcoma, Kaposi's sarcoma, oteogenic and other sarcoma, renal cell tumor, glioblastoma multiforma, malignant melanomas, epidermoid carcinomas.
[00268] In embodiments, the methods comprise administering to the subject a pharmaceutical composition described herein, such as a pharmaceutical composition comprising an isolated or recombinant histone polypeptide or a fragment thereof.
[00269] In embodiments, the pharmaceutical composition can be administered in a therapeutically effective amount.” The term “therapeutically effective” can refer to that amount of the composition sufficient to treat a disease and/or ameliorate one or more causes or symptoms of a disease or disorder. Amelioration, for example, only requires a reduction or alteration, not necessarily elimination. As used herein, the terms “therapeutically effective amount” “therapeutic amount” and “pharmaceutically effective amount” are synonymous. One of skill in the art can readily determine the proper therapeutic amount.
[00270] The term “administration” or “administering” can refer to the act of physically delivering, e.g., via injection or an oral route, a substance as it exists outside the body into a patient, such as by oral, subcutaneous, mucosal, intradermal, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, disorder or condition, or a symptom thereof, is being treated therapeutically, administration of the substance can occur after the onset of the disease, disorder or condition or symptoms thereof. Prophylactic treatment involves the administration of the substance at a time prior to the onset of the disease, disorder or condition or symptoms thereof.
[00271] In embodiments, one or more additional molecules can be administered (i.e., coadministered) to the subject. The phrase “co-administration” can refer to simultaneous and sequential administration of two or more compounds or compositions. An appropriate time course for sequential administration can be chosen by the physician, according to such factors as the nature of a patient's illness, and the patient's condition. Non-limiting examples of additional molecules that can be administered to the subject together with a composition as described herein can comprise conventional therapeutics, such as conventional cancer therapeutics.
[00272] Convention cancer therapeutics include, but are not limited to, chemotherapeutic agents, cytokines, chemokines, and radiation therapy. The majority of chemotherapeutic drugs can be divided into alkylating agents, antimetabolites, an thracy dines, plant alkaloids, topoisomerase inhibitors, and other anti-tumor agents. These drugs can affect cell division or DNA synthesis and function in some way. Additional therapeutics include monoclonal antibodies and the new tyrosine kinase inhibitors e.g. imatimb mesylate, which directly targets a molecular abnormality in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors).
[00273] Representative chemotherapeutic agents include, but are not limited to cisplatm, carboplatin, oxaliplatm, mechlorethamine, cyclophosphamide, chlorambucil, vincristine, vinblastine, vinorelbine, vindesme, taxoi and derivatives thereof, irmotecan, topotecan, amsacrine, etoposide, etoposide phosphate, temposide, epipodophyllotoxins, trastuzuinab (HERCEPT1N®), cetuximab, and rituximab, hevaeizumah, and combinations thereof.
[00274]
[00275] Other Embodiments
[00276] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. [00277] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
[00278] Examples are provided herein to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
EXAMPLE 1
[00279] H3 is a secreted protein
• BirA system and othoganal validation [00280] Histone 3 Secretion is autophagy dependent
• FBS supplementation suppresses H3 secretion
• Bafilomycin A1 but not Brefeldin A suppresses H3 secretion
• sgATG7 and sgBCNl impair H3 secretion
• Rapamycin Increase H3 secretion
[00281] Tool compounds to study protein trafficking
• Brefeldin A-inhibitor of “classical” ER to Golgi Protein Secretion o inhibits Sec7-type-GTP-exchange factors thereby preventing coat protein assembly on ER to Golgi protein transport vesicles
• Bafilomycin Al- inhibitor of Cellular Autophagy o Inhibits V-ATPase, a membrane spanning proton pump that acidifies the lysosome
[00282] Secreted H3 is post-translationally modified
• PTM Western Blot • Mutant Data
[00283] Secreted H3 is taken up by destination cells
• Dual Fluorescence Assays (FACS and Microscopy)
• Dual Fluorescence Transwell Assay
• Myr-Recipient and RFP-Donor
• Halo Microscopy
CreReporter assay Data
EXAMPLE 2
[00284] Han, H., Yang, J., Chen, W., Li, Q., Yang, Y., and Li, Q. (2019). A comprehensive review on histone-mediated transfection for gene therapy. Biotechnol Adv 37, 132-144. [00285] Hariton-Gazal, E., Rosenbluh, J., Graessmann, A., Gilon, C., and Loyter, A. (2003). Direct translocation of histone molecules across cell membranes. J Cell Sci 116, 4577-4586. [00286] Ryser, H.J., and Hancock, R. (1965). Histones and basic polyamino acids stimulate the uptake albumin by tumor cells in culture. Science 150, 501-503.
[00287] Wagstaff, K.M., Glover, D.J., Tremethick, D.J., and Jans, D.A. (2007). Histone- mediated transduction as an efficient means for gene delivery. Mol Ther 15, 721-731.
[00288] Peeters, Janneke GC, et al. "Transcriptional and epigenetic profiling of nutrient- deprived cells to identify novel regulators of autophagy." Autophagy 15.1 (2019): 98-112. [00289] US20210061867A1 [00290] US20160130329A1
EXAMPLE 3
[00291] Nucleic Acid Sequence of Histone H3 Fused to Cre Recombinase
[00292] ATG (bolded and underlined) methionine start codon [00293] TCCAAGGAC (Bolded) - Human Histone H3 Sequence (408 Nucleotides - 136
Amino Acids
[00294] LINKER (italicized) - 23 Amino Acids ( 69 Nucleotides) same as the one used between mEmerald and H3
[00295] CRE Recombinase (underlined)
[00296] tag stop codon added (bolded and underlined)
[00297] ACCCAGCTTTCTTGTACAAAGTGGTCCCC= attB2 sequence(SEQ ID NO: 42)
Figure imgf000079_0001
Figure imgf000080_0001
[00298]
[00299] Protein Acid Sequence H3 Fused to Cre Recombinase
Figure imgf000080_0002
[00300]
[00301] H3 fused to mEmerald Nucleic Acid Sequences
[00302] ATGGTGAGC (underlined) - mEmerald Sequence (717 Nucleotides - 239 Amino
Acids') [00303] ACCTACGGC (Bold and underlined) mEmerald Chromophore (TYG);
[00304] A206K = AAG (bold and underlined!
[00305] TCCAAGGAC - (bold) Human Histone H3 Sequence (408 Nucleotides - 136 Amino Acids)
[00306] LINKER (italicized) - 23 Amino Acids ( 69 Nucleotides) between mEmerald and H3 [00307] ATG - (bold and underlined) Methionine Start Codon [00308] TAA - (bold and underlined) Stop Codons
Figure imgf000081_0001
Figure imgf000082_0001
[00309] H3 fused to mEmerald Protein Sequence
Figure imgf000082_0002
[00310]
[00311] Histone 3 Fused to mCherry Nucleic Acid Sequence
[00312] GCCACGATG (underline) - mCherry Sequence (708 Nucleotides - 236 Amino Acids AY6782641
[00313] ATGTACGGC - (bold and underline) mCherry Chromophore (MYG) [00314] TCCAAGGAC (bolded) - Human Histone H3 Sequence (408 Nucleotides - 136 Amino Acids)
[00315] LINKER -( italicized) 23 Amino Acids ( 69 Nucleotides) between mCherry and
H3
[00316] ATG (bold and underline) - Methionine Start Codon
[00317] TAA - (bold and underline) Stop Codon
Figure imgf000083_0001
[00318]
[00319] Histone 3 Fused to mCherry Protein Sequence
Figure imgf000083_0002
Figure imgf000084_0001
EXAMPLE 4
[00320] Horizontal Transfer of Histone H3 by Mammalian Cells [00321] Histones are small, highly basic, nuclear proteins that serve as structural elements to condense DNA into chromatin and regulate its accessibility1. Although histones released from dying cells can act as extracellular signaling molecules, without wishing to be bound by theory, all intracellular histone molecules have originated from the cell in which they reside2 6. Here we show that histone H3 is horizontally transferred between viable cells. Using an ER-targeted biotin ligase to detect secreted proteins7·8, we serendipitously discovered that histone H3 is selectively secreted by autophagic cells relative to histones H2A, H2B, and H4. Specific H3 posttranslational modifications (PTMs) are enriched or depleted on secreted H3 relative to intracellular H3, indicating that specific PTMs dictate H3’s ability to be secreted or the information it conveys to neighboring cells. Remarkably, we found that secreted H3 can enter the nuclei of recipient cells in an autophagy -independent and cell contact-independent manner ex vivo and in vivo. These findings have implications for cell-cell communication during nutrient deprivation, hypoxia, and perhaps other forms of cellular stress, and for the ability to deliver macromolecules across cell membranes.
[00322] To identify new secreted biomarkers controlled by the VHL tumor suppressor gene, we introduced the BirA biotin ligase fused to an ER localization signal (ER-BirA7·8), or the corresponding empty expression vector (EV), into 786-0 human VHL-I- clear cell renal carcinoma cells (ccRCCs) that reexpress the VHL in the presence of Doxy cy cline (DOX) (FIG. 47 panels a & b, and FIG. 51, panels a & b). In experiments we detected the secreted proteins IGFBP3, VEGF, and Clusterin in the conditioned media (CM) of the ER-BirA cells, but not the EV cells, after capture on streptavidin beads and immunoblot analysis (FIG. 47, panel c). Reexpression of VHL suppressed IGFBP3 and VEGF, and induced Clusterin9. This assay was sensitive, based on the detection of VEGF (present at <10 nM in CM10 11) and specific, based on the failure to detect abundant intracellular proteins such as vinculin, b- Actin, b-Tubulin, COX IV, Golgin 97, Calreticulin and Lamin A/C in the CM (FIG. 47, panels c and d).
[00323] Surprisingly, we detected the apparent independent secretion of histone
Figure imgf000085_0001
H3 (H3) with this assay (FIG. 47, panel e). This was specific because histone H4 (H4), histone H2A (H2A), and histone H2B (H2B) were not detected in the CM. This was not a BirA artefact, because we likewise detected H3, but not H4, H2A, or H2B, in parental 786-0 CM that was concentrated using a spin column or trichloroacetic acid (TCA) precipitation (FIG. 47, panel f). No H3 was detected in unconditioned cell culture media (FIG. 51, panel c). In the experiments that follow, TCA precipitation was used to concentrate the CM unless otherwise specified. H3 secretion was confirmed using two independent H3 antibodies (FIG. 47, panel g) and in multiple cell lines including RCC4, UMRC2, 769-P, and A498 ccRCC lines, MCF7 and MDA-MB-231 human breast carcinoma cells, U20S human osteosarcoma cells, B16 mouse melanoma cells, and HOG human oligodendroglioma cells (FIG. 51, panels d-f). We also observed secretion of the oncohistone H3K27M12 13 by SF8628 diffuse intrinsic pontine glioma cells (FIG. 51, panel f). The secreted H3 is indicated to be monomeric, as determined by non-denaturing Native Gel Electrophoresis of spin column concentrated media (FIG. 51, panels g & h), and was sensitive to trypsin and proteinase K digestion, indicating that it was not encapsulated in an extracellular vesicle14 (FIG. 51, panels I & j).
[00324] The specific detection of H3 without other histones (nor other abundant intracellular proteins) strongly indicated against non-specific release of H3 by dying cells. Moreover, the posttranslational modifications of the secreted H3 were not representative of the total pool of H3, with a clear bias toward histone hypermethylation and against acetylation (FIG. 47, panels h & i). Acetylation of H3 lysine 14 (H3K14) was an exception, as it was dramatically increased on secreted H3 relative to bulk H3 (FIG. 47, panel h). This indicates that specific PTMs govern H3 secretion or its downstream consequences. Further, mutating H3 lysine 14 to arginine blocked secretion, whereas mutating it to glutamine to mimic lysine acetylation enhanced secretion (FIG. 47, panel j), and mutating lysine 27 to methionine, corresponding to a canonical oncohistone mutation, increased H3 secretion (FIG. 51, panel k).
[00325] To ask how H3 is secreted, we treated RCC4 VHL-/- ccRCC cells with brefeldin A, which blocks canonical protein secretion15, or bafilomycin Al, which blocks autophagy -dependent secretion16 17. Brefeldin A blocked the secretion of IGFBP3, but did not block the secretion of H3 (FIG. 48, panel a). In contrast, bafilomycin Al blocked the secretion of H3, but not IGFB3 (FIG. 48, panel a), as did CRISPR/Cas9-based elimination of ATG7 or Beclin 1, which are required for autophagy17"19 (FIG. 48, panels b-e).
[00326] Our CM experiments above were performed in synthetic, serum-free, media to reduce media complexity. Low serum promotes autophagy19·20. Notably, we did not detect apoptosis after 48 hours of serum starvation (FIG. 52, panels a-f). Conversely, apoptosis- inducing agents did not promote the selective release of H3 (FIG. 52, panel g). We confirmed that readdition of serum to the culture medium suppressed H3 secretion (FIG. 48, panel f), whereas induction of autophagy by treatment with rapamycin21 or exposure to hypoxia22 promoted H3 secretion (FIG. 48, panels g-i, and FIG. 52, panel h). Therefore, H3 undergoes autophagy-dependent secretion. Prompted by this finding, we confirmed that LC3B, which undergoes autophagy-dependent secretion, was likewise captured in streptavidin pulldowns of CM from BirA-ER expressing cells (FIG. 52, panel i) and that its secretion was likewise suppressed by bafilomycin Al, but not brefeldin A (FIG. 52, panel j).
[00327] To monitor the fate of secreted H3, we created 786-0 cells that express H3 fused to mEmerald, a brightly fluorescent monomeric variant of green fluorescent protein, or mCherry, mixed them 1:1, grew them in serum-poor media, and performed live cell FACS. Remarkably, double-positive mEmerald/mCherry cells appeared within 48 hours (FIG. 49, panels a & b, and FIG. 53, panel a). This was specific, because double-positive cells were virtually absent when H2A, H2B, or H4 fusion cells, were tested in parallel (FIG. 49, panels a & b, and FIG 53, panel a). Similar results were observed using a Doxycycline-inducible expression system in 786-0 and 293T cells (FIG. 53, panels b-f) and when autophagy was stimulated by hypoxia rather than by growth in low serum (FIG. 54, panels a, b).
[00328] In a complementary set of experiments, we co-cultured 293T human embryonic kidney cells expressing either H3-mCherry or H4-mCherry with RCC4 cells expressing a membrane-bound GFP (myr-GFP). Once again, we observed double-positive cells by FACS with the fluorescent H3 fusion protein cells, but not the H4 fusion protein cells (FIG. 49, panels c & d). Using live cell fluorescence microscopy, we observed rare green cells with red nuclear inclusions that corresponded to mitotic chromosomes after Hoechst dye staining (FIG. 49, panel c). Importantly, uptake of H3, in contrast to secretion of H3, was unaffected by loss of ATG7 or Beclin 1 (FIG. 55, panels a-d).
[00329] The apparent transfer of H3 did not require contact between donor and recipient cells based on transfer experiments in which filtered donor-conditioned media was added to separately plated recipient cells (FIG. 55, panels a-d) and based on transwell experiments in which recipient and donor cells were separated by a 3 pm filter (FIG. 49, panels e & f). To complement our fluorescence-based assays, we harvested the CM from RCC4 cells expressing FLAG epitope-tagged H3 or H4 and added it to RCC4 cells expressing BirA (Turbo-ID Variant7) fused to a nuclear localization signal (NLS) or the corresponding EV. 48 hours later we lysed the recipient cells, captured proteins on streptavidin beads, and did immunoblot assays. We detected Flag-H3, but not Flag-H4 in the streptavidin pulldowns from NLS-BirA extracts, but not EV extracts (FIG. 49, panels g & h). As additional controls, we confirmed that abundant non-nuclear recipient cell proteins were not biotinylated in this assay (FIG. 49, panel h).
[00330] In analogous experiments with SF8628 “donor” cells, which endogenously express H3K27M (see herein), and 786-0 cells “recipient” expressing BirA-NLS, we detected H3K27M in the streptavidin pulldowns from NLS-BirA extracts, but not EV extracts (FIG. 55, panels e & f), indicating endogenously produced oncohistones can be transferred to non-oncohistone producing cells.
[00331] Single-molecule, live-cell imaging of 293T cells expressing of H3 or H4 fused to HaloTag confirmed secretion of H3, but not H4, after serum-starvation (FIG. 56, panels a & b). Without wishing to be bound by theory, the low basal H3-HaloTag and H4-HaloTag detected in the media without serum starvation by this highly sensitive technique reflects the low basal levels of amphisomal secretion of nucleosomes reported in unstressed cells23. We co-cultured 293T cells expressing H3-HaloTag or H4-HaloTag with RCC4 cells expressing H2B-mEmerald in the absence of serum. 48 hours later, 15-20% of the H2B-mEmerald RCC4 cells contained H3-HaloTag, as determined by single molecule imaging, with -80% of the protein in the cytoplasm and -20% in the nucleus (FIG. 56, panels c-g). In contrast, transfer of H4 was virtually undetectable. Without wishing to be bound by theory, uptake into the nucleus would increase upon dissolution of the nuclear membrane during mitosis. [00332] Encouraged by these results, we cocultured 786-0 cells expressing H3 or H4 fused to Cre recombinase with mouse tex.loxP.EG cells24 that conditionally express GFP after Cre-mediated excision of a Lox-Stop-Lox cassette (FIG. 49, panel I, and FIG. 57, panel a). We first confirmed that both the H3-Cre and H4-Cre fusions could recombine a GFP- based reporter in cotransfection experiments (FIG. 57, panels b-c). In contrast, only H3-Cre, but not H4-Cre, induced GFP expression in the recipient cell coculture experiments, indicating that H3, but not H4, can deliver a functional payload to the nucleus (FIG. 49, panels j & k). Similar results were obtained with a “color switch reporter25” in which Cre- recombination switches expression from a red fluorescent protein (RFP) to GFP (FIG. 49, panel n).
[00333] Consistent with the secretion results (FIG. 47, panel j), mutating H3K14 to arginine blocked transfer, whereas mutating H3K14 to glutamine to mimic lysine acetylation enhanced transfer of both H3-Cre and fluorescently -tagged Histone 3 (FIG. 57 and FIG. 58). [00334] To ask whether horizontal transfer of H3 can take place in vivo, we mixed 786-0 cells expressing H3-Cre or H4-Cre 1:1 with 786-0 cells with the color switch reporter and grew them subcutaneously in nude mice (FIG. 50, panel a). Notably, the Cre expressing cells grew more slowly than the color switch cells, without wishing to be bound by theory, because targeting Cre recombinase to chromatin impairs cellular fitness. Five weeks later, we removed palpable tumors, dissociated the tumor cells, and monitored recombination by FACS and immunoblot analysis for GFP-positive cells. The H3-Cre, but not H4-Cre, cells promoted recombination of the color switch reporter (FIG. 50, panels b-d, and FIG. 59, panel a). Remarkably, H3-Cre-dependent recombination was also detectable when the Cre donor cells and color switch recipient cells were grown as separate tumors on opposite flanks (FIG. 50, panels e-h, and FIG. 59, panel b). Finally, we detected circulating H3, but not circulating H4, in non-tumor bearing mice that were fasted to promote autophagy 26,27 (FIG. 50, panels I
&j)·
[00335] Histones released by dying cells can elicit danger signals by interacting with Toll-like receptors on cell surfaces and contribute to the pathogenesis of diseases such as septic shock2,5. Our findings indicate that autophagy causes the specific secretion of H3 in a form that can cross cell membranes and enter the nuclei of other cells. Our data indicate that certain PTMs are enriched or depleted on secreted H3 compared to the bulk H3 population, indicating that specific PTMs influence H3 secretion, uptake, or the information conveyed to recipient cells. H3K14 acetylation, for example, appears to promote H3 secretion. We will validate that the transfer of H3 from donor to recipient cells influences cell behavior, such as through incorporation into chromatin or by anon-canonical function. Transfer of histone H3 can reflect an ancient signaling mechanism that predated the emergence of cytokines, growth factors and hormones.
[00336] Solid tumors invariably contain hypoxic cells and therefore, without wishing to be bound by theory, secrete H3. We will validate that circulating H3 contributes to any of the systemic manifestations of cancer, such as cachexia and hypercoagulability, and that they can serve as diagnostic or prognostic biomarkers. Our findings that an oncohistone can cross cell membranes also indicate that certain oncohistones could play paracrine roles in transformation.
[00337] Exogenous histones can cross cell membranes under certain experimental conditions and can enhance the transfection efficiency of DNA, although the physiological relevance of these findings was previously unclear28"33. Delivering macromolecules across cell membranes has been a holy grail in drug discovery, although previous protein transduction methods based on positively charged peptides and proteins such as the Antennapedia protein homeodomain (penetratin), HIV TAT, and histones, have not proven robust34. Our finding that the H3 secreted by autophagic cells can be used to deliver a protein cargo indicates that histone-based protein transduction can be optimized further by understanding the structure-function relationships underlying this phenomenon, including the importance of specific H3 PTMs, together perhaps with directed evolution.
[00338] References Cited in this Example
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2 Xu, J. el al. Extracellular histones are major mediators of death in sepsis. Nature medicine 15, 1318-1321 (2009).
3 Remijsen, Q. el al. Dying for a cause: NETosis, mechanisms behind an antimicrobial cell death modality. Cell Death & Differentiation 18, 581-588 (2011).
4 Yipp, B. G. & Kubes, P. NETosis: how vital is it? Blood 122, 2784-2794 (2013).
5 Silk, E., Zhao, EL, Weng, H. & Ma, D. The role of extracellular histone in organ injury. Cell death & disease 8, e2812-e2812 (2017).
6 Bemardes, N. E. & Chook, Y. M. Nuclear import of histones. Biochemical Society Transactions 48, 2753-2767 (2020).
7 Branon, T. C. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nature biotechnology 36, 880-887 (2018).
8 Droujinine, I. A. et al. Proteomics of protein trafficking by in vivo tissue-specific labeling. Nature Communications 12, 2382, doi:10.1038/s41467-021-22599-x (2021).
9 Nakamura, E. et al. Clusterin is a secreted marker for a hypoxia-inducible factor- independent function of the von Hippel-Lindau tumor suppressor protein. The American journal of pathology 168, 574-584 (2006). 10 Iliopoulos, O., Levy, A. P., Jiang, C., Kaelin, W. G. & Goldberg, M. A. Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proceedings of the National Academy of Sciences 93, 10595-10599 (1996).
11 Siemeister, G. et al. Reversion of deregulated expression of vascular endothelial growth factor in human renal carcinoma cells by von Hippel-Lindau tumor suppressor protein. Cancer Research 56, 2299-2301 (1996).
12 Nacev, B. A. et al. The expanding landscape of ‘oncohistone’ mutations in human cancers. Nature 567, 473-478 (2019).
13 Koch, L. Cancer genetics: Oncohistone pathology explained. Nat Rev Genet 17, 375, doi:10.1038/nrg.2016.71 (2016).
14 Leidal, A. M. et al. The LC3-conjugation machinery specifies the loading of RNA- binding proteins into extracellular vesicles. Nature cell biology 22, 187-199 (2020).
15 Donaldson, J. G., Finazzi, D. & Klausner, R. D. Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature 360, 350-352 (1992).
16 Yoshimori, T., Yamamoto, A., Moriyama, Y., Futai, M. & Tashiro, Y. Bafilomycin Al, a specific inhibitor of vacuolar-type H (+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. Journal of Biological Chemistry 266, 17707- 17712 (1991).
17 Cavalli, G. & Cenci, S. Autophagy and protein secretion. Journal of Molecular Biology 432, 2525-2545 (2020).
18 Yu, L. et al. Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science 304, 1500-1502 (2004).
19 Mizushima, N. Autophagy: process and function. Genes & development 21, 2861-
2873 (2007). 20 Kaizuka, T. et al. An autophagic flux probe that releases an internal control.
Molecular cell 64, 835-849 (2016).
21 Jung, C. H., Ro, S.-H., Cao, J., Otto, N. M. & Kim, D.-H. mTOR regulation of autophagy. FEBS letters 584, 1287-1295 (2010).
22 Mazure, N. M. & Pouyssegur, J. Hypoxia-induced autophagy: cell death or cell survival? Current opinion in cell biology 22, 177-180 (2010).
23 Jeppesen, D. K. et al. Reassessment of exosome composition. Cell 177, 428-445. e418 (2019).
24 Wadia, J. S., Stan, R. V. & Dowdy, S. F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nature medicine 10, 310-315 (2004).
25 Zomer, A. et al. In vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior. Cell 161, 1046-1057 (2015).
26 Komatsu, M. et al. Impairment of starvation-induced and constitutive autophagy in Atg7 -deficient mice. Journal of Cell Biology 169, 425-434 (2005).
27 Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T. & Ohsumi, Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Molecular biology of the cell 15, 1101-1111 (2004).
28 Hariton-Gazal, E., Rosenbluh, J., Graessmann, A., Gilon, C. & Loyter, A. Direct translocation of histone molecules across cell membranes. Journal of Cell Science 116, 4577- 4586 (2003).
29 Fritz, J. D., Herweijer, H., Zhang, G. & Wolff, J. A. Gene transfer into mammalian cells using histone-condensed plasmid DNA. Human gene therapy 7, 1395-1404 (1996).
30 Ryser, H. J.-P. & Hancock, R. Histones and basic polyamino acids stimulate the uptake of albumin by tumor cells in culture. Science 150, 501-503 (1965). 31 Schwartz, A. The Effects of Histones and Other Poly cations on Cellular Energetics: I.
MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION. Journal of Biological Chemistry 240, 939-943 (1965).
32 Han, H. et al. A comprehensive review on histone-mediated transfection for gene therapy. Biotechnology advances 37, 132-144 (2019).
33 Wagstaff, K. M., Glover, D. J., Tremethick, D. J. & Jans, D. A. Histone-mediated transduction as an efficient means for gene delivery. Molecular Therapy 15, 721-731 (2007).
34 Lonn, P. & Dowdy, S. F. Cationic PTD/CPP-mediated macromolecular delivery: charging into the cell. Expert opinion on drug delivery 12, 1627-1636 (2015).
EXAMPLE 5
[00339] Methods
[00340] Cell Culture
[00341] 786-0, 769-P, A498, U20S, MDA-MB-231, MCF7, and HEK293T cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) + 10 % Fetal Bovine Serum (FBS) + 1 % Penicillin/Streptomycin (Pen/Strep). RCC4 cells. UMRC-2 cells were maintained in DMEM + 10% FBS + 1% Pen/Strep. B16-F10 mouse melanoma cells maintained in DMEM + 10% FBS + 1% Pen/Strep. HOG cells (human oligodendroglioma line) were maintained in DMEM + 10% FBS + 1% Pen/Strep. SF8628 maintained in DMEM + 10% FBS + 1% Pen/Strep. Tex.loxP.EG cells2 were maintained in Roswell Park Memorial Institute (RPMI) Medium + 10% FBS + 1% Pen/Strep.
[00342] 786-0 cells with doxycycline-inducible VHL expression have been previously descrived3 and were maintained in DMEM + 10% FBS + 1 % P/S + 800 pg/mL G418. After lentiviral integration of pLX-304-BirA*-G3-ER (or empty vector control) 786-0 cells expressing doxycycline-inducible VHL and BirA*-G3-ER were maintained in DMEM + 10% FBS + 1 % P/S + 800 pg/mL G418 + 5 pg/mL blasticidin. [00343] 786-0, RCC4 and 293T cells expressing histone fusion cDNA ORFs or BirA-
NLS cloned into the pLenti-gate-PGK-Puro backbone were maintained in DMEM + 10%
FBS + 1% Pen/Strep + 1 pg/mL puromycin. 786-0 or 293T cells expressing doxycycline- inducible fluorescence histone fusion ORF cDNAs in the plix_403 (Addgene # 41395) were maintained in DMEM + 10% FBS + 1% Pen/Strep + 1 pg/mL puromycin. RCC4 stably expressing Tet-myr-GFP or expressing LC3-GFP were maintained in DMEM + 10% FBS + 1% Pen/Strep + 1 pg/mL puromycin.786-0 and RCC4 cells expressing the “color switch” Cre-reporter (pLV-CMV-LoxP-DsRed-LoxP-eGFP) were and maintained in DMEM + 10% FBS + 1% Pen/Strep + 1 pg/mL puromycin.
[00344] Chemicals
[00345] Biotin (Sigma- Aldrich) was prepared to a stock solution of 200 mM in autoclaved, milliQ filtered water and was diluted in cell culture medium to the indicated final concentration. Doxy cy dine (Sigma- Aldrich) was prepared to a stock solution of 1 mg/mL in sterile phosphate-buffered saline (PBS) and was diluted in cell culture medium to the indicated final concentration. Propidium Iodide (PI) (ThermoFisher) was diluted in cell culture medium to the indicated final concentration. Brefeldin A (Selleck Chemicals) was prepared as a stock solution of 10 pM in DMSO and was diluted in cell culture medium to the indicated final concentration. Bafilomycin A1 (Cell Signaling Technologies) was prepared as a stock solution of 100 mM in DMSO and was diluted in cell culture medium to the indicated final concentration. Rapamycin was prepared as a stock solution of 1 mM in DMSO and was diluted in cell culture medium to the indicated final concentrations. Hoechst 33342 (ThermoFisher) was first diluted to a lOx stock (10 pg/mL in Opti-MEM) and then added to the cell culture medium to a final concentration of 1 pg/mL.
[00346] Plasmids [00347] pCMV 6-H3 -FLAG and pCMV6-H3K27M-FLAG have been previously described4. mEmerald-H3-32 (Addgene #54115), mCherry-H3-23 (Addgene #55058), mEmerald-H4-23 (Addgene #54117) mCherry H4-23 (Addgene #55060), plix_403 (Addgene #41395), pLX304 (Addgene #25890), and 3xHA-TurboID-NLS_pCDNA3 (Addgene #107171), pLV -CMV -LoxP-DsRed-LoxP-eGFP (Addgene #65726), Tet-myr-GFP (Addgene #83468) pMRX-IP-GFP-LC3-RFP-LC3AG (Addgene #84572), were obtained from Addgene Plasmid Repository. pLenti-CRISPR-V2-Hygro has been previously described and was cloned in the Kaelin Laboratory by swapping out the puromycin resistance gene in pLenti- CRISPR-V2 for a hygromycin resistance gene5. Generation of pLenti-CRISPR-V2-Hygro with gRNA sequences targeting ATG7 and Beclin 1 (BECN1) are described in detail below. pLenti-Efla-gate-PGK-Puro has been previously described6·7 and the cloning of the histone fusion ORF cDNAs into pLenti-Efl a-gate-PGK-Puro and pbx_403, cloning of plx304- BirA*-G3-ER, and cloning of pLenti-Efla- BirA-NLS -PGK-Puro are all described in detail below.
[00348] Cloning of CRISPRJCas9 Plasmids
[00349] pLenti-CRISPR-V2-Hygro was digested with BsmBI (New England Biolabs, no. R0580) or FastDigest Esp 31 (Life Technologies, no. FD0454) for 30 minutes (min) at 37°C, and the resulting linearized vectors were gel-purified. Oligonucleotides encoding 4 independent sgRNAs targeting ATG7 or Becbn-1 from the Brunello sgRNA Library8 with corresponding Bsm BI/Esp 31 overhangs added to facilitate ligation were synthesized by Integrated DNA Technologies . Oligonucleotides were annealed using 0.15 nmol of each sense and antisense oligonucleotides. The oligonucleotides were heated at 95°C for 4 min and allowed to slowly cool to room temperature. Annealed oligonucleotides were then diluted 1 : 100 in nuclease-free water and ligated into the linearized vectors using T4 ligase in a 4 hour (h) incubation at room temperature. A 2-pL aliquot of the ligation mixture was then transformed into 20 pL of HB101 chemically competent F coli cells (Promega, no. L2011).
Plasmids from ampicillin-resistant colonies were isolated by QIAprep Spin Plasmid Miniprep
Kit (Qiagen, no. 27106) and validated by DNA sequencing. The sgRNA oligonucleotides used for editing (including Bsm BI/Esp 31 overhangs) are: sgATG7 #1 F: 5 ’C ACCGCCAGAAAATATTCCCCGGTG3 ’ sgATG7 #1 R: 5’AAACCACCGGGGAATATTTTCTGGC 3’ sgATG7 #2 F: 5’CACCGCTCTTGTAAATACCATCTGT3’ sgATG7 #2 R: 5’AAACACAGATGGTATTTACAAGAGC 3’ sgATG7 #3 F: 5’ C ACCGCTTGAAAGACTCGAGTGTGT3 ’ sgATG7 #3 R: 5’AAACACACACTCGAGTCTTTCAAGC 3’ sgATG7 #4 F: 5’ CACCGTCCTACTTTAGACTTGGACA3’ sgATG7 #4 R: 5’AAACTGTCCAAGTCTAAAGTAGGAC 3’ sgBECNl #1 F: 5’ CACCGAGTGGC AGAAAATCTCGAGA3 ’ sgBECNl #1 R: 5 ’AAACTCTCGAGATTTTCTGCCACTC 3’ sgBECNl #2 F: 5’ CACCGCTGTGC ATTCCTCACAGAGT3 ’ sgBECNl #2 R: 5’AAACACTCTGTGAGGAATGCACAGC 3’ sgBECNl #3 F: 5’ CACCGGAAGGTTGCATTAAAGACGT3’ sgBECNl #3 R: 5’AAACACGTCTTTAATGCAACCTTCC 3’ sgBECNl #4 F: 5’ CACCGGGAAGAGACTAACTCAGGAG 3’ sgBECNl #4 R: 5’AAACCTCCTGAGTTAGTCTCTTCCC 3’
[00350] Cloning of Histone Fusion Protein and Histone-FLAG Open Reading Frame
(ORF) expression Vectors
[00351] The histone fusion ORF cDNA were cloned into pLenti-Efla-gate-PGK-Puro, for constitutive expression, or plix_403, for doxycycline-inducible expression, using the gateway cloning method9. First, the histone fusion ORF cDNAs were amplified by PCR. [00352] The histone3-mEmerald ORF cDNA was PCR amplified from mEmerald-H3-
32 (Addgene #54115) using the following primers (which include the attb sequences for gateway cloning):
F : 5 ’ GGGGAC A AGTTTGTAC A AA AAAGC AGGCTTC ATGGCTCGTACTAA AC AG AC AGCT3 ’ and R:
5 ’ GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACTTGTACAGCTCGTCCATGCC3 ’ .
[00353] The histone3-mCherry ORF cDNA was PCR amplified from mCherry-H3-23 (Addgene #55058) using the following primers (which include the attb sequences for gateway cloning): F: 5’
GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCTCGTACTAAACAGACAGCT 3’ and R:
5 ’ GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACTTGTACAGCTCGTCCATGCC3 ’ .
[00354] The histone4-mEmerald ORF cDNA was PCR amplified from mEmerald-H4- 23 (Addgene #54117) using the following primers (which include the attb sequences for gateway cloning): F:
5 ’ GGGGAC AAGTTTGTAC A AAA AAGC AGGCTTC ATGTCTGGCCGCGGC A AAGGCGGG3 ’ and R:
5 ’ GGGACC ACTTTGTAC AAG AA AGCTGGGTCTTACTTGTAC AGCTCGTCC ATGCC3 ’ .
[00355] The histone4-mCherry ORF cDNA was PCR amplified from mCherry H4-23 (Addgene #55060) using the following primers (which include the attb sequences for gateway cloning): F: 5’
GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGGCCGCGGCAAAGGCGGG3’ and R: 5’ GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACTTGTACAGCTCGTCCATGCC3 ’ .
[00356] The H3-FLAG ORF was PCR amplified from pCMV6-H3.3 (Origene RC202257) using primers : F: 5’G
GGGGAC A AGTTTGTAC AAA AAAGC AGGCTTC ATGGCTCGTACAAAGCAGACTGCCC3 ’ and R: 5’ GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAAACCTTATCGTCGTCATCC3’ [00357] After confirmation of successful PCR amplification by agarose gel electrophoresis, the PCR products were purified using the QiaQuick PCR Purification Kit (Qiagen). The PCR products were then recombined into pDONR-223 using BP Clonase reactions. Each BP Clonase reaction consisted of 1 pL of the purified PCR product, 100 ng of pDONR-223 (at 100 ng/pL), 1 pL BP Clonase (ThermoFisher) and 2 pL of IX Tris-EDTA (TE) Buffer (pH 7), and was incubated for 2 h at room temperature.
[00358] The H2A-mEmerald, H2A-mCherry, H2B-mEmerald, H2B-mCherry, H4- FLAG, H3-Cre, H4-Cre, H3-HaloTag and H4-HaloTag histone fusion ORF cDNA were synthesized as dsDNA gBlocks by Integrated DNA Technologies. The synthetic dsDNA fragments contained a 5’ attbl site, a 3’ attb2 site, and introduced the same 23 amino acid linker used in the H3 and H4 mEmerald and mCherry fusions described above between the indicated histones and fusion partners.
[00359] To recombine the gBlocks into pDONR-223, each BP clonase reaction consisted of 10 pL of the gBlock resuspended to 10 ng/pL in IX TE, 100 ng of pDONR-223 (at 100 ng/pL), 1 pL BP Clonase (ThermoFisher). All BP Clonase reactions were incubated at room temperature for 2 hours, and then 2 pL of each clonase reaction was transformed into HB101 chemically competent E. coli. Successful transformants were selected by growth on Lysogeny Broth (LB)-agar supplemented with 50 pg/mL spectinomycin plates. Drug- resistant colonies were picked and then grown overnight at 37 °C in 2 mL of LB + 100 pg/mL spectinomycin. Bacterial cultures were pelleted, and plasmid DNA was then isolated using Qiaprep Spin Miniprep Kit (Qiagen). The pDONR-223 vectors containing the histone fusion ORF cDNAs were verified by Sanger Sequencing using the M13F and M13R sequencing primers.
[00360] The histone fusion ORF cDNAs were then recombined from pDONR-223 into the lenti viral expression vectors pLenti-Efla-gate-PGK-Puro3·5·6 or plix_403 (Addgene #41395) using LR Clonase (ThermoFisher). Each LR Clonase recombination reaction consisted of 1 pL pDONR-223 containing the histone fusion ORF at 100 ng/pL. 1 pL of the lentiviral expression vector at 100 ng/pL, 2 pL of LR clonase, and 7 pL of IX TE Buffer (pH 7). 2 pL of Each LR reaction was transformed into HB101 chemically competent E.coli. Successful transformants were selected by growth on LB-agar supplemented with 50 pg/mL kanamycin. Drug-resistant colonies were then picked and grown overnight at 30 °C in 2mL of LB + 50 pg/mL kanamycin. 1 mL of each bacterial culture was pelleted to isolate plasmid DNA using QIAprep Spin Miniprep Kit (Qiagen), and 1 mL of culture was saved at 4 °C. Correct clones were identified by Sanger sequencing, and then the 1 mL of saved culture was used to inoculate a 50 mL culture used to prepare sufficient quantities of the vector for lentiviral production. The plasmid prep from the 50 mL culture was again verified by Sanger Sequencing.
[00361] Si te-Directed Mutagenesis
[00362] Point mutant Histone-Fusion ORF cDNAs (H3K14R, H3K14Q and H3K27M) were generated using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs). For the FLAG-Tagged H3 point mutants, site-directed mutagenesis was performed on pCMV6-H3.3 and verified by Sanger Sequencing. To make point mutants in the H3-mEmerald, H3- mCherry and H3-Cre ORFs, the mutations were introduced into the pDONR-223 construct and verified by Sanger sequencing. The mutant ORF cDNA was then recombined into a lentiviral expression vector using an LR recombination reaction (cloning methodology described above). The final plasmid preps were confirmed using Sanger sequencing before generation of lentiviruses. The primers used for site-directed mutagenesis were as follows: H3K14R-F: 5 CACCGGCGGTCGAGCGCCACGCA3’,
H3K14R-R: 5 ’GATTTCCGAGCTGTCTGTTTAGTACGAGCC3 ’
H3K14Q-F: 5’ CACCGGCGGTCAAGCGCCACGCA3’ H3K14R-R: 5’ GATTTCCGAGCTGTCTGTTTAGTACGAGCC ATG3 ’ .
H3K27M-F: 5’ GGCTGCTCGCATGAGCGCGCCGG3 ’
H3K27M-R: 5’ TTGGTAGCCAGCTGCTTGCGTGG3’.
[00363] Cloning of the BirA *-G3-ER
[00364] BirA-ER cells were created by stably expressing the BirA*-G3-ER, a highly promiscuous and active variant of BirA fused to a KDEL tag for ER localization; this strategy has been previously successfully employed to localize BirA-G3* the the ER in vitro and in vivo10 11. pLX-304-BirA-G3*-ER was cloned recombining the BirA-G3*-ER ORF cDNA10 from a pDONR gateway entry clone into pLX304 using the gateway cloning methodology detailed above.
[00365] Cloning of BirA-NLS
[00366] BirA-NLS cells were created by stably expressing the Turbo-ID variant of BirA fused to a SV-40 NLS signal and his strategy has been previously successfully employed to localize Turbo ID to the nucleus11. The Turbo ID variant of BirA, does not require a “target sequence” to biotinylate a substrate and has been extensively used in proximity ligation experimetns12. We amplified the TurboID-NLS ORF cDNA from 3xHA- TurboID-NLS_pCDNA3 (Addgene #107171) using the primers:
F: 5 ’ GGGGACAAGTTTGTAC AAAAAAGC AGGCTATGTACCCGTATGATGTTCCGGA T3’
R:
5 ’ GGGGACCACTTTGTACAAGAAAGCTGGGTTCAC ACCTTCCTCTTCTTCTTGGG3 ’ We then cloned the successfully amplified PCR ORF cDNA into pLenti-Efla-gate-PGK- Puro using the gateway cloning methodology detailed above.
[00367] Lentiviral Production and Infection [00368] HEK293FT Cells were plated at a density of 2.5 x 106 cells per 10-cm dish. 48 h later, they were transfected with 10 pg of lentiviral transfer vector, 8 pg of psPAX2, and 2 pg of pMD2.G. 72 h after transfection, the media was collected and filtered through a 45 pm filter, and stored at 4 °C. Fresh growth medium (DMEM + 10% FBS + 1% Pen/Strep) was then added to virus-producing HEK293FT cells. The next day, 96 h post transfection, the media was again collected and filtered through a 45 pM filter, combined with the media collected at 72 h post transfection, and supplemented with polybrene at 8 pg/mL, before addition to the target cells for infection.
[00369] 24 h before infection, target cells to be infected were plated at a density of 1 x
106 cells per dish in 10-cm dishes. For infection growth media was removed, the cells were washed once with PBS, and then 3.5 mL of virus containing media was added per dish.
Target cells were incubated with the virus containing media for 48 h at 37 °C. The virus- containing media was then removed, and fresh growth media was added for 24 h. Next, selective media was added: for infection with viruses derived from the pLenti-Efla-PGK- Puro backbone and for viruses from the plix_403 backbone 1 pg/mL puromycin was added, for infection with viruses derived from the pLX304 backbone 5 pg/mL blasticidin was added, and for CRISPR/Cas9 knockouts using viruses derived from the pLenti-CRISPR-V2-Hygro backbone5 500 pg/mL hygromycin B was added. For infection of cells with pLV-CMV- LoxP-DsRed-LoxP-eGF (“color switch” Cre-Reporter) or, tet-myr-GFP, or 1 pg/mL puromycin was added.
[00370] After approximately one week of selection, stable expression of fluorescent histone fusion ORFs in target cells was confirmed by live-cell fluorescence microscopy after staining with 1 pg/mL Hoechst 33342. Expression of histone-Cre fusion proteins was confirmed by anti-Cre immunoblot, and knockout of target genes was confirmed by immunoblot. All the cells generated by stable lentiviral infection, including cells expressing histone fusions and cells that had undergone CRISPR/Cas9-based gene editing, were maintained as polyclonal pools in drug selection media.
[00371] Retroviral Production and Infection
[00372] HEK293FT cells were plated at a density of 2.5 x 106 cells per 10-cm dish. 48 h later, they were transfected with 10 pg of pMRX-IP-GFP-LC3-RFP-LC3AG (the retroviral transfer vector encoding GFP-LC3 and RFP-LC3AG) and 10 pg of pCL-Ampho (Novus Bio). Virus production and infection protocols were then identical to the lentiviral protocols detailed above. After infection, stable cells were generated by selection with lug/mL puromycin, and successful selection was confirmed by live-cell fluorescence microscopy visualizing GFP-LC3.
[00373] Spin Column Concentration of Conditioned Media
[00374] 786-0 cells were plated at a density of 4 x 106 cells per dish in 15-cm dishes in DMEM + 10% FBS + 1% Pen/Strep. 48 h later the growth media was removed, and the cells were washed twice with PBS prior to the addition of 15 mL of fresh Opti-MEM. 48 h later, the conditioned Opti-MEM media was collected, spun at lOOg for 5 min at 4 °C to gently pellet any cells that might have dislodged during collection, and transferred to a 3 kDa cut-off filter column made of regenerated cellulose (Millipore UFC900324). The column was spun at 3800g for 15 min at room temperature. The flow through was then discarded and the column was spun for an additional 20 min at room temperature, after which the remaining flow through was discarded and the column retentate was removed by pipetting and kept as the spin column concentrated conditioned media sample. To prepare spin column concentrated conditioned media samples for Native (Non-Denaturing Page), the centrifugation was carried out at 4°C, instead of at room temperature.
[00375] Trichloroacetic Acid (TCA) Precipitation of Conditioned Cell Culture Media [00376] Cells were plated in 6-well culture dishes at a density of 2 x 105 cells per well. 48 h later, the cells were washed twice with PBS and 1.5 ml Opti-MEM was then added per well. After 48 h of culture in Opti-MEM, the media was collected and spun down at lOOg for 5 min at 4 °C to gently pellet any cells that might have dislodged during collection. After centrifugation, 1 mL of the conditioned media was transferred to a fresh tube. Next, 250 pL of 100% w/v TCA was added to conditioned media and the sample was incubated on ice for 30 min. Next, the samples were centrifuged at 17,000g for 15 min at 4 °C to pellet the precipitated protein, and the supernatant was removed. The sample pellets were then washed three times with 800 pL acetone followed by a 5 min centrifugation at 17,000g and 4 °C. After removal of the final acetone wash, the pellets were air dried for 5 min at room temperature and then further dried for 5 min at 95 °C to drive off any residual acetone. “Media” samples indicated on Immunoblot figures are TCA precipitated protein from 1 mL of conditioned Opti-MEM resuspended in 20 pL of lx Sample Buffer (unless otherwise indicated) and assayed by immunoblot along “Cells” samples (10 pg of whole cell lysate, prepared as indicated).
[00377] Immunoblot analysis
[00378] Cells grown in 12-well, 6-well, or 10-cm tissue culture dishes were washed once with lx phosphate-buffered saline (PBS) and then detached by incubation with 0.05% Trypsin for 5 min at 37 °C. The cells were resuspended in DMEM + 10% FBS + 1% Pen/Strep, transferred to conical tubes, pelleted by centrifugation at lOOg for 5 min, washed once with ice-cold PBS, and then transferred to a 1.5-ml microcentrifuge tube. Cells were then resuspended in Cell Lysis Buffer (50 mM Tris, 250 mM NaCl, 1% Igepal, 0.1% SDS, 5 mM EDTA, 10 mM Na2P2Ch, 10 mM NaF), briefly vortexed, and incubated for 30 min on ice. The lysates were then clarified by centrifugation at 17,000g for 10 min at 4 °C and transferred to a new microcentrifuge tube. The protein concentration of the whole cell extracts was quantified using a BCA Protein Assay (Thermo Fisher Scientific, no. PI23227). After denaturing for 5 min at 95 °C in lx Sample Buffer (lx Sample Buffer: 2.2% SDS, 11% glycerol, 100 mM dithiothreitol, bromophenol blue), 10 pg of protein was loaded per lane for whole cell lysate samples. Samples derived from conditioned media were processed and loaded as described below. Samples were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes using a Trans-Blot Turbo (Bio-Rad, no. 1704155). Membranes were blocked by incubation in 5% milk/tris-buffered saline (TBS)/0.1% Tween 20 (TBS-T) for 1 hour at room temperature, washed three times with TBS-T (5 min per wash), and then probed with primary antibody diluted in either TBS-T + 5% Bovine Serum Albumin (BSA) or TBS-T + 5% milk as indicated, and incubated overnight at 4 °C with gentle agitation. After primary antibody incubation, membranes were washed three times with TBS-T (5 min per wash) and then incubated with 1 :5000 horseradish peroxidase (HRP)-conjugated secondary antibody in 5% milk/TBS-T [goat anti-mouse immunoglobulin G (IgG) (Thermo Fisher Scientific, no. 31430) or goat anti-rabbit IgG (Thermo Fisher Scientific, no. 31460)] for 1 hour at room temperature with gentle agitation. Bound antibodies were then detected with enhanced chemiluminescence Western blotting reagents (Thermo Fisher Scientific, no. WBKLS0500) or Super-Signal West Pico (Thermo Fisher Scientific, no. PI34078).
[00379] The primary antibodies used were: rabbit a-VHL (1:1000 in TBS-T + 5%
BSA; Cell Signaling, no. 68547), rabbit a-HIF-2a (1: 1000 in TBS-T + 5% BSA; Cell Signaling, no. 59973), mouse a-vinculin (1:10,000 in TBS-T + 5% milk; Sigma, no. V9131), rabbit a-NDRGl (1:1000 in TBS-T + 5% BSA; Cell Signaling, no. 5196), rabbit a-IGFBP3 (1:10,000 in TBS-T + 5% BSA; Cell Signaling no. 25864), rabbit a-VEGF (1:1000 in in TBS-T + 5% milk; Abeam no. ab46154), rabbit a-Myc (HRP conjugate) (1:100 in TBS-T + 5% BSA; Cell Signaling no. 14038), rabbit a-b-Actin (HRP conjugate, 1:2000 in TBS-T + 5% BSA; Cell Signaling no.12620), rabbit a-b-Tubulin (HRP conjugate, 1:2000 in TBS-T + 5% BSA; Cell Signaling no.5346), rabbit a-COX IV (HRP conjugate, 1:2000 in TBS-T + 5% BSA; Cell Signaling no.5247), rabbit a-Golgin 97 (1:2000 in TBS-T + 5% BSA; Cell Signaling no. 13192), rabbit a-Calreticulin (1:2000 in TBS-T + 5% BSA; Cell Signaling no. 12238), rabbit a-Histone H3 (“Ab #1”, HRP conjugate, 1:5000 in TBS-T + 5% BSA; Cell Signaling no. 12648), rabbit a-Histone H3 (“Ab #2”, 1:5000 in TBS-T + 5% BSA; Abeam no. abl8521), rabbit a-Histone H4 (1:5000 in TBS-T + 5% BSA; Cell Signaling no. 13919), rabbit a-Histone H2A (1:2000 in TBS-T + 5% BSA; Cell Signaling no. 12349), rabbit a- Histone H2B (1:5000 in TBS-T + 5% BSA; Cell Signaling no. 12364), mouse a-Lamin A/C (1:5000 in TBS-T + 5% BSA; Cell Signaling no. 4777), rabbit a-H3K14Ac (1:1000 in TBS-T + 5% BSA; Cell Signaling no. 7627), rabbit a-H3K4Ac (1:2000 in TBS-T + 5% milk; Abeam no. ab 176799), rabbit a-H3K9Ac (1:5000 in TBS-T + 5% BSA; Cell Signaling no. 9649), rabbit a-H3K18Ac (1:2000 in TBS-T + 5% BSA; Cell Signaling no. 13998), rabbit a- H3K27Ac (1:2000 in TBS-T + 5% BSA; Cell Signaling no. 8173), rabbit a-H3K36Ac (1:2000 in TBS-T + 5% BSA; Cell Signaling no. 27683), rabbit a-H3K56Ac (1:2000 in TBS- T + 5% BSA; Cell Signaling no. 4243), rabbit a-H3K4me2 (1:2000 in TBS-T + 5% BSA;
Cell Signaling no. 9725), rabbit a-H3K4me3(l:2000 in TBS-T + 5% BSA; Cell Signaling no. 9751), rabbit a-H3K9me2 (1:2000 in TBS-T + 5% BSA; Cell Signaling no. 4658), rabbit a- H3K9me3 (1:5000 in TBS-T + 5% BSA; Cell Signaling no. 13969), rabbit a-H3K27me2 (1:2000 in TBS-T + 5% BSA; Cell Signaling no. 9728), rabbit a-H3K27me3 (1:2000 in TBS- T + 5% BSA; Cell Signaling no. 9733), rabbit a-H3K36me2 (1:2000 in TBS-T + 5% BSA; Cell Signaling no. 2901), rabbit a-H3K36me3(l:2000 in TBS-T + 5% BSA; Cell Signaling no. 4904), rabbit a-FLAG (1:10,000 in TBS-T + 5% BSA; Cell Signaling no. 14793), rabbit a-MMP2 (1:5000 in TBS-T + 5% BSA; Cell Signaling no. 87809), rabbit a-LC3B (1:2000 in TBS-T + 5% BSA; Cell Signaling no. 3868), rabbit a-H3K27M (“Ab #1”, 1:2000 in TBS-T + 5% milk; Abeam no. abl90631), rabbit a-H3K27M (“Ab #2”, 1:2000 in TBS-T + 5% BSA;
Cell Signaling no. 74829), rabbit a-Caspase 3 (1:1000 in TBS-T + 5% BSA; Cell Signaling no. 14220), rabbit a-Cleaved Caspase-3 (1:1000 in TBS-T + 5% BSA; Cell Signaling no. 9664), rabbit a-ATG7(l:2000 in TBS-T + 5% BSA; Cell Signaling no. 8558), rabbit a-P62 (1:2000 in TBS-T + 5% milk; abeam no. ab56416), rabbit a-Beclin-1 (1:1000 in in TBS-T + 5% BSA; Cell Signaling no. 3738), rabbit a-P04S6-Kinase (1:1000 in in TBS-T + 5% BSA; Cell Signaling no. 9234), rabbit a-Cre-Recombinase (1:2000 in in TBS-T + 5% BSA; Cell Signaling no. 15036), and rabbit a-GFP-tag (HRP conjugate 1:2000 in in TBS-T + 5% BSA; Cell Signaling no. 2037).
[00380] Biotin Labeling and Recovery of Secreted Proteins
[00381] 786-0 cells (with doxycycline-inducible VHL expression) expressing BirA-
G3-ER, a highly promiscuous variant of Turbo ID fused to a KDEL tag for ER localization10 11, or the corresponding empty vector, were plated at a density of 2 x 106 cells per dish in 15-cm dishes in DMEM + 10% FBS + 1% Pen/Strep + 5 pg/mL Blasticidin and 500 pg/mL G418, with or without 2 pg/mL doxy cy cline. 96 hours later, at which point the cells were approximately 90 % confluent, the cells were split and replated at a density of 4 x 106 cells per dish in 15-cm dishes in DMEM + 10% FBS + 1% Pen/Strep + 5 pg/mL Blasticidin and 500 pg/mL G418, with or without 2 pg/mL doxy cy cline. 48 h later, the growth media was removed, and the cells were washed 2 times with PBS prior to the addition of 15 mL Opti-MEM without selective antibiotics, but supplemented with 20 pM biotin (with or without 2 pg/mL doxy cy cline). 48 h later, the conditioned Opti-MEM media was collected, spun at lOOg for 5 min at room temperature to gently pellet any cells that might have dislodged during collection, and filtered through a 45 pm syringe filter. Next the conditioned media was transferred to a 3 kDa cut-off filter column made of regenerated cellulose (Millipore UFC900324). The column was spun at 3800g for 15 min at room temperature. The flow through was then discarded and the column was spun for an additional 20 min, after which the remaining flow through was again discarded. The column was then washed twice with 10 mL of PBS per wash, each followed by centrifugation at 3800g for 15 min at room temperature and subsequent discarding of the flow through. The column retentate was removed by pipetting and kept for subsequent analysis. In pilot experiments these two PBS wash steps were found to be necessary to remove residual biotin that was retained and interfered with subsequent capture and detection of biotinylated proteins.
[00382] To enrich for biotinylated proteins, magnetic streptavidin beads (ThermoFisher 65002) were first blocked by incubation in TBST containing 1% BSA with gentle rocking for 1 hour at room temperature. 30 pL of beads were added to 300 pi of concentrated conditioned media. The samples were then gently rocked for 1 hr at room temperature. The beads were then washed twice with Cell Lysis Buffer and twice with IX TBST. After the final wash, the beads were resuspended in 40 pL of IX Sample Buffer and heated for 10 min at 95 °C to elute the proteins. For western blot analysis 10 pg of protein from whole cell lysate or 20 pL of eluate from streptavidin pulldowns were loaded per lane. For streptavidin HRP blots, nitrocellulose membranes were blocked for 1 h in TBST + 5% BSA and then incubated with Streptavidin HRP reagent (Thermo Fisher), diluted 1:2500 in TBST + 5% BSA, overnight with gentle rocking at 4 °C. Bound HRP was detected with enhanced chemiluminescence Western blotting reagents (Thermo Fisher Scientific, no. WBKLS0500).
[00383] Native (Non-Denaturing) PAGE
[00384] 20 pL of Spin-Column concentrated media (centrifuged at 4°C) was prepared in Native Gel Loading Buffer (2.5X TBE + 50% Glycerol, 0.1% Bromophenol Blue) that was supplemented with 1 pL of Native Page 5% G-250 Sample Additive (ThermoFisher) per sample and loaded into NativePAGE 3-12% Bis Tris gels. The concentrated conditioned media was run alongside the indicated amounts of recombinant nucleosomes or recombinant histone 3.3. Electrophoresis was conducted in aXCell SureLock Mini-Cell Electrophoresis System (ThermoFisher) using Dark Blue Cathode Buffer containing 0.02 % G-250 (ThermoFisher) in the Upper (inner) Buffer Chamber and NativePAGE Running Buffer (Anonde Buffer) in the Lower (outer) chamber at 150 V for 30 min at 4 °C. After 30 min, the Dark Blue Cathode Buffer (ThermoFisher) containing 0.02 % G-250 in the Upper Buffer Chamber was replaced with the Light Blue Cathode Buffer containing 0.002 % G-250 (ThermoFisher) and the electrophoresis was resumed at 150 V for approximately 2 h at 4 °C, at which point the dye front approached the end of the gel.
[00385] Prior to transfer, the PVDF membrane was incubated in 100% ethanol for 2 min and then incubated in TurboTransfer Buffer (BioRad) for 2 min. Proteins were transferred using the BioRad TurboTransfer system. After transfer, the PVDF membrane was destained by washing three times with 50% Methanol (10 min with gentle rocking per wash), once in TBST-T + 0.5% SDS (5 min with gentle rocking per wash), and three times in TBS-T (5 min with gentle rocking per wash). Membranes were blocked by incubation in 5% milk in TBS-T gently rocking for 1 hour at room temperature and immunoblotted as indicated. [00386] Protease Protection Assays
[00387] 786-0 cells were plated at a density of 4 x 106 cells per dish in 15-cm dishes in DMEM + 10% FBS + 1% Pen/Strep. 48 h later the growth media was removed, and the cells were washed twice with PBS prior to the addition of 15 mL of fresh Opti-MEM. 48 h later, the conditioned Opti-MEM media was collected, spun at lOOg for 5 min at room temperature to gently pellet any cells that might have dislodged during collection. Next, 1 mL of conditioned media was aliquoted per experimental condition. Trypsin was added to a final concertation of 100 pg/ml, with or without 1% TritonX-100, and incubated for 30 min at 4 °C. Alternatively, Proteinase K was added to a final concentration of 200 pg/mL, with or without 1% TritonX-100, and incubated for 1 h at 37 °C. After the incubation, TCA was added to a final concentration of 25% w/v and the samples were processed using the TCA precipitation protocol described above.
[00388] Propidium Iodide (PI) Uptake, andAnnexin V / PI Co-Staining [00389] 786-0 cells were plated at a density of 5 x 104 cells per well in 12-well dishes in DMEM + 10% FBS + 1% Pen/Strep. 48 h later, the growth media was removed, and the cells were washed twice with PBS prior to the addition of 1.5 mL of fresh Opti-MEM, Opti- MEM + FBS, or OPTIMEM + the indicated concentration of puromycin per well. 48 h later, all attached and floating cells were collected and pelleted by centrifugation at lOOg for 5 min at 4 °C. The pellets were then washed once with ice-cold PBS. For PI uptake experiments, the cells were resuspended in ice cold FACS buffer (PBS + 1% BSA) supplemented with PI at a final concentration of 1 pg/mL. For Annexin V and PI Co-Staining experiments, cells were prepared using the Dead Cell Apoptosis Kit with Annexin V FITC and PI for flow cytometry (ThermoFisher) per the manufacturer’s protocol. Briefly, cells were resuspended in lx Annexin V Binding Buffer (ThermoFisher) and incubated with 1 pg/mL PI and 5% FITC Annexin V Component A solution (ThermoFisher). The cells were then analyzed by live-cell flow cytometry at the Dana-Farber Flow Cytometry Core, analyzing 10,000 cells per sample using a BD LSRFortessa flow cytometer with BD FACSDiva software. FCS files were exported and analyzed using FlowJo software.
[00390] FLAG-tagged Histone Secretion Assay
[00391] U20S cells were plated at a density of 2 x 105 cells per well in 6-well dishes in DMEM + 10% FBS + 1% Pen/Strep. 24 h later, the growth media was removed, and the cells were washed twice with PBS prior to the addition of 1.5 mL of fresh Opti-MEM. 24 h later, the cells were transfected with the 1 pg Plasmid DNA of WT pCMV-H3-FLAG, empty vector control, or the indicated point mutant using 1 pL Lipofectamine 2000 per well. 24 h after transfection, the conditioned culture media was collected, TCA precipitated, and immunoblotted.
[00392] Green-Red Dual-Fluorescence Assays for Histone Transfer
[00393] To co-culture constitutively expressing H3-mEmerald and H3-mCherry cells at a 1:1 ratio, 786-0, RCC4 or 293T cells expressing pLenti-Efla-H3-mEmerald-PGK-Puro or pLenti-Efla-H3-mCherry-PGK-Puro growing at 70-90% confluence in 10-cm dishes were detached with 0.05% trypsin for 5 min at 37 °C and resuspended in 10 mL DMEM + 10 % FBS + 1% Pen/Strep. Cells were counted and resuspended at a density of 2.5 x 104 H3- mEmerald expressing cells per mL and 2.5 x 104 H3-mCherry expressing cells per mL (a total density of 5 x 104 cells per mL). The cells were thoroughly mixed by gentle vortexing (3 times for 5 seconds each) and inversion (3 times) and then plated by adding 1 mL of cell suspension per well in 12-well dishes. In parallel, single-color controls (monocultures of H3- mEmerald or H3-mCherry expressing cells) were plated at 5 x 104 cells per well using identical protocols. 48 h later, the growth media was removed, and the cells were washed 2 times with PBS prior to the addition of 1.5 mL/well of Opti-MEM without selective antibiotics. The same procedure was followed for analogous experiments cells expressing H2A, H2B, H4, or H3 mutants fused to either mEmerald or mCherry.
[00394] For co-culture of mCherry histone fusion expressing cells with Tet-myr-GFP expressing cells, 1:1 co-cultures were plated and carried out as above and supplemented with lpg/mL doxy cy dine. Stable, pooled, myr-GFP cells were generated by lentiviral integration of Tet-myr-GFP (Addgene # 83468) using the lentiviral production and infection methodology described above.
[00395] For co-culture experiments with doxycycline-inducible histone fusions, cells were plated grown in DMEM + 10% FBS + 1 % Pen/Strep. 2 pg/mL doxy cy dine was or was not added at the time of plating for co-culture assays and maintained when the growth media was switched to Opti-MEM.
[00396] After 48h of co-culture in Opti-MEM or the indicated media, cells were analyzed by live-cell flow cytometry at the Dana-Farber Flow cytometry Core. 10,000 cells were analyzed per sample using a BD LSRFortessa flow cytometer with BD FACSDiva software. FCS files were exported and analyzed using FlowJo software. Gating was established based on the single-color control samples. As an additional control, single-color control samples were mixed immediately before FACS analysis (no time in co-culture). [00397] Cell Labeling and Preparation for Single-Molecule Imaging [00398] RCC4-mCherry, RCC4-H2B-GFP cells, 293T-H3-HaloTag and 293T-H4- HaloTag cells were cultured in the imaging media, which was prepared by supplementing FluoroBrite medium (Invitrogen) with 10% FBS, 1 mM glutamax and 0.1 mM nonessential amino acids.
[00399] To collect the conditioned media from H3-HaloTag and H4-HaloTag 293T “donor” cells, cells were first washed three times with lx PBS and then cultured in Opti- MEM (Thermofisher) for 24 hours before adding O.lnM JF646-HTL for HaloTag labeling. After culture for 24h in JF646-HTL supplemented Opti-MEM, the conditioned media was harvested and centrifuged at l,500g for 10 mins.
[00400] To assay the abundance of H3-HaloTag and H4-HaloTag molecules in the conditioned media, we plated conditioned media onto ultra-clean cover glasses for 30 mins before Total Internal Reflection Fluorescence (TIRF) imaging.
[00401] To image uptake of H3-HaloTag and H4-HaloTag by RCC4 cells in the absence of the “donor” cells, RCC4 cells were plated on ultra-clean cover glasses and were incubated with the hybrid media consisting of 50% conditioned and 50% imaging media for 24 h. To image the transfer of H3-HaloTag from “donor” cells to RCC4 “recipient” cells in
- Ill - the same dish, 293T “donor” and RCC4 “recipient” cells were plated on ultra-clean cover glasses with a ratio of 1 : 10 in the imaging media. After culturing overnight, cells were washed with three times with PBS (5 min per wash) prior to being incubated with Opti-Mem for 48 hours. The labeling was performed in the imaging media with InM JF646-HTL for 30 mins.
[00402] Before imaging, the cells were washed 3 times (10 min per wash) with imaging media, and then 100 ng/ml Hoechst dye added to delineate cell nuclei. Then, the cover glasses were transferred to Attofluor metal holders (Thermofisher) and mounted onto the microscope for highly inclined and laminated optical sheet (HILO) imaging.
[00403] Single-Molecule Imaging
[00404] Single-molecule imaging was used to assess accumulation of H3/H4 HaloTag molecules on glass surface and transfer of H3-HaloTag into RCC4 cells. Specifically, imaging was performed on a Nikon Eclipse TiE Motorized Inverted microscope equipped with a 100X Oil-immersion objective lens (Nikon, N.A. = 1.49), four laser lines (405/488/561/642), an automatic TIRE illuminator, a perfect focusing system, a tri-cam splitter, three EMCCDs (iXon Ultra 897, Andor) and Tokai Hit environmental control (humidity, 37 °C, 5% CO2). A band mirror (405/488/561/633; BrightLine quad-band bandpass filter) was used to reflect the lasers into the objective lens. Proper emission filters (Semrock) were placed in front of the 3 cameras for simultaneous imaging of Hoechst, mCherry and JF646. JF646 labelled H3-HaloTag and H4-HaloTag molecules were imaged at 50 Hz using a 642 nm laser with the excitation intensity of ~ 1000 W/cm2. 5000 frames were collected for each field of view.
[00405] Single-Molecule Localization, Tracking and Diffusion Analysis
[00406] For single-molecule localization and tracking, the spot localization (x,y) was obtained through 2D Gaussian fitting based on MTT algorithms13. The localization and tracking parameters are listed in the Table 1. Diffusion coefficients were calculated from tracks with at least 5 consecutive frames by the MSDanalyzer14 with a minimal fitting R2 of
0 8
Figure imgf000114_0001
[00407] Transwell Assays
[00408] 786-0 cells expressing H3-mEmerald, H3-mCherry, H4-mEmerald or H4- mCherry were plated at a density of 5 x 104 cells per “donor” (upper) well and 5 x 104 cells per “recipient” (lower) well in 24-well, 3 mM membrane transwell plates (MilliporeSigma CLS3398) in DMEM + 10% FBS + 1% Pen/Strep. 24 h later, the growth media was removed, and the cells were washed twice with PBS prior to the addition of 1.5 mL of fresh Opti-MEM per well. After 48 h in Opti-MEM, the cells in the “recipient” well were detached by incubation with 0.05% trypsin for 5 min at 37 °C and then resuspended in growth media. The cells were then analyzed by live-cell flow cytometry at the Dana Farber Flow Cytometry Core, analyzing 10,000 cells per sample using a BD LSRFortessa flow cytometer with BD FACSDiva software. FCS files were exported and analyzed using FlowJo software. Gating was established based on the single-color control samples. As an additional control, singlecolor control samples were mixed immediately before FACS analysis (no time in co-culture). [00409] Transfer ofH3 to the Nucleus ofBirA-NLS Cells
[00410] “Donor” 786-0 cells expressing H3-FLAG were seeded at a density of of 4 x
106 cells per dish in 15-cm dishes in DMEM + 10% FBS + 1% Pen/Strep + 5 pg/mL Blasticidin and 500 pg/mL G418, with or without 2 pg/mL doxy cy dine. 48 h later, the growth media was removed, and the cells were washed 2 times with PBS prior to the addition of 15 mL Opti-MEM. 48 h later, the conditioned Opti-MEM media was collected, spun at lOOg for 5 min at room temperature to gently pellet any cells that might have dislodged during collection. 1 mL of conditioned media was added to “recipient” 786-0 cells expressing BirA-NLS (TurboID-NLS), 24 h after “recipient” cells were plated in 12-well dishes at 2.5 x 104 cells per well. After 24 of culture in conditioned medium, 20 mM biotin was added. After another 24 h, the cells were washed 4 times with PBS, collected, detached with 0.05% trypsin for 5 min at 37 °C, resuspended in growth media, pelleted by centrifugation for 5 min at lOOg, washed twice more with PBS, and lysed with 200 pL Cell Lysis Buffer for 30 min on ice. The lysates were then clarified by centrifugation at 17,000g for 10 min at 4° C and transferred to new tubes. At this point, 20 pL of lysate was taken to be used as input. To enrich for biotinylated proteins, magnetic streptavidin beads (ThermoFisher 65002) were first blocked by incubation in TBST containing 1% BSA with gentle rocking for 1 hour at room temperature. 30 pL of beads were added to 180 pi of “recipient” cell lysate and the samples were gently rocked for 1 hr at room temperature. The beads were washed twice with Cell Lysis Buffer, once with 2 M Urea, twice more with Cell Lysis Buffer, and then twice with TBS-T. After the final wash, the beads were resuspended in 20 pL of IX Sample Buffer and heated for 10 min at 95° C to elute the proteins.
[00411] To co-culture “donor” SF8628 cells endogenously expressing the H3K27M oncohistone, with “recipient” 786-0 cells expressing BirA-NLS (TurboID-NLS) at a 1:1 ratio, cells growing at 70-90% confluence in 10-cm dishes were first detached with 0.05% trypsin for 5 min at 37 °C, and resuspended in 10 mL DMEM + 10 % FBS + 1% Pen/Strep. Next, the cells were counted and resuspended together at a density of 2.5 x 104 SF8628 cells per mL and 2.5 x 104 BirA-NLS expressing 786-0 cells per mL (a total density of 5 x 104 cells per mL). After thoroughly mixing by gentle vortexing (3 times for 5 seconds each) and subsequent inversion (3 times), the cells were plated by adding 1 mL of cell suspension per well in 12-well dishes. HOG cells which secrete H3, but do not express the H3K27M oncohistone, were identically co-cultured with BirA -NLS 786-0 cells as a control. After 24 h in co-culture the growth media was removed and the cells were then washed twice with PBS before the addition of 1.5 ml of fresh Opti-MEM per well. 24 h after adding the Opti- MEM media to the co-cultures, 20 mM biotin was added. After another 24 h, the cells were washed 4 times with PBS, collected, detached with 0.05% trypsin for 5 min at 37 °C, resuspended in growth media, pelleted by centrifugation for 5 min at lOOg, washed twice more with PBS, and lysed with 200 pL Cell Lysis Buffer for 30 min on ice. The lysates were then clarified by centrifugation at 17,000g for 10 min at 4° C and transferred to new tubes. At this point, 20 pL of lysate was taken to be used as input. To enrich for biotinylated proteins, magnetic streptavidin beads (ThermoFisher 65002) were first blocked by incubation in TBST containing 1% BSA with gentle rocking for 1 hour at room temperature. 30 pL of beads were added to 180 pi of “recipient” cell lysate and the samples were gently rocked for 1 hr at room temperature. The beads were washed twice with Cell Lysis Buffer, once with 2 M Urea, twice more with Cell Lysis Buffer, and then twice with TBS-T. After the final wash, the beads were resuspended in 20 pL of IX Sample Buffer and heated for 10 min at 95° C to elute the proteins.
[00412] In vitro Cre-Recombinase Reporter Assays
[00413] To co-culture H3-Cre expressing cells with “color switch” Cre-reporter cells at a 1:1 ratio, H3-Cre expressing RCC4 cells (pLenti-Efla-H3-Cre-PGK-Puro) and “color switch” Cre-reporter expressing RCC4 cells (pLV-CMV-LoxP-DsRed-LoxP-eGFP), were grown to 70-90% confluence in 10-cm dishes, detached with 0.05% trypsin for 5 min at 37 °C, resuspended in 10 mL DMEM + 10 % FBS + 1% Pen/Strep + 1 pg/mL Puromycin. Cells were then resuspended at a density of 2.5 x 104 H3-Cre expressing cells and of 2.5 x 104 “color switch” Cre-reporter cells per mL (a total density of 5 x 104 cells per mL). After thoroughly mixing by gentle vortexing (3 times for 5 second each) and inversion 3 times, cell were plated by adding 1 mL of cell suspension per well in 12-well dishes. 48 h later the growth media was removed, and the cells were washed twice with PBS prior to the addition of 1.5 mL/well of Opti-MEM.
[00414] “Donor” cells expressing H4-Cre (pLenti-Efla-H4-Cre-PGK-Puro) were cocultured using identical protocols and included as controls. Co-culture experiments for “donor” cells expressing histone 3 Cre fusion ORFS with the indicated point mutants (H3K14R, H3K14Q, and H3K27M in pLenti-Efla-H3-Cre-PGK-Puro) were carried out identically as described herein.
[00415] After 48h of co-culture in Opti-MEM or in the indicated media, the cells were analyzed by live-cell flow cytometry at the Dana-Farber Flow Cytometry Core, analyzing 10,000 cells per sample using a BD LSRFortessa flow cytometer with BD FACSDiva software. FCS files were exported and analyzed using FlowJo software. Gating was established based monoculture of reporter cells, and as an additional control Cre-fusion expressing cells and reporter cells were mixed immediately before FACS analysis (no time in co-culture).
[00416] To co-culture H3-Cre (pLenti-Efla-H3-Cre-PGK-Puro) expressing cells with tex.loxP.EG cells2 (exhausted T-Cells which grow in suspension, harboring a lox-stop-lox GFP cassette) at a 1 : 1 ratio, H3-Cre expressing cells were plated at a density of 5 x 104 per well in 12-well dishes in DMEM + 10% FBS + 1% FBS + 1 pg/mL Puromycin. Log-phase tex.loxP.EG cells were pelleted and resuspended in Opti-MEM at a density of 5 x 104 Cells/mL. After washing the H3-Cre cells twice with PBS, 1 mL of the tex.loxP.EG cell suspension was added per well. 48 h later, the Opti-MEM media containing the tex.loxP.EG cells growing in suspension was collected and analyzed by live-cell flow cytometry at the Dana Farber Flow Cytometry Core, analyzing 10,000 cells per sample using a BD LSRFortessa flow cytometer with BD FACSDiva software. FCS files were exported and analyzed using FlowJo software. Cells expressing H4-Cre (pLenti-Efla-H4-Cre-PGK-Puro) were co-cultured using identical protocols and included as controls.
[00417] 50/50 Histone-Cre and Cre-Reporter Co-Xenografts
[00418] 786-0 Cells expressing H3-Cre (pLenti-Efla-H3-Cre-PGK-Puro) or H4-Cre
(pLenti-Efla-H4-Cre-PGK-Puro) as well as 786-0 cells expressing the “color switch” Cre- reporter (pLV-CMV-LoxP-DsRed-LoxP-eGFP) were cultured to 90% confluence in DMEM+ 10% FBS + 1% Pens/Strep + 1 pg/ml puromycin. After detaching the cells with 0.05% trypsin at 37 °C for 5 min, the cells were counted, and 50 million H3-Cre cells were resuspended with 50 million “color switch” cells in 1 mL of PBS + 0.2% FBS. The cell suspension was gently vortexed to mix, and then 10 million cells (injection volume of 100 pL) were injected subcutaneously into the flank of approximately 10-week-old female NCr mice (Taconic, #NCRNU-F) anesthetized by isoflurane inhalation. As controls, H4-Cre 50/50 xenografts were prepared similarly. 10 mice were injected per experimental group. All procedures followed approved IACUC protocols, and mice were monitored daily for changes in weight, activity, and food and water intake. 5 weeks later, once palpable tumors had formed, the animals were sacrificed, and the tumor was processed for flow cytometry and immunoblot analysis.
[00419] To dissociate the tumors for flow cytometry, a piece of the tumor was cut off with a razor blade, then thoroughly minced with a razor blade in a 10-cm cell culture dish to break up and manually dissociate the tumor cells. 5 mL of 0.05% Trypsin was added to the cells and then the mixture was incubated at 37 °C for 10 min. 5 mL of DMEM + 10% FBS + 1% Pen/Strep was then added, and the suspension was vortexed (3 times for 20 seconds each). After gentle vortexing, the sample was passed through a 35 pm cell strainer 3 times, and the suspension was centrifuged at 200g to pellet the dissociated cells. The pellet was resuspended in 1 mL DMEM + 10% FBS + 1% Pen/Strep and passed through a 35 pm cell strainer a final time before flow cytometry analysis. The cells were analyzed by live-cell flow cytometry at the Dana Farber Flow Cytometry Core, analyzing 20,000 cells per sample using a BD LSRFortessa flow cytometer with BD FACSDiva software. FCS files were exported and analyzed using FlowJo software.
[00420] In parallel, a piece of tumor was taken for immunoblot analysis. This piece of the tumor was cut off with a razor blade, then thoroughly minced with a razor blade in a 10- cm cell culture dish to break up the tumor and manually dissociate the tumor cells. Next, the cells were collected in 500 pL of Cell Lysis Buffer and incubated on ice for 30 min with gentle vortexing every 5 min. Immunoblotting was then performed using the protocol detailed above. The data shown represent 2 true biological replicates using different cohorts of mice and independently generated “donor” and “recipient” cells.
[00421] Dual Flank Histone-Cre and Cre-Reporter Xenografts [00422] 786-0 Cells expressing H3-Cre or H4-Cre as well as 786-0 cells expressing the “color switch” Cre-reporter (pLV-CMV-LoxP-DsRed-LoxP-eGFP) were cultured to 90% confluence in DMEM+ 10% FBS + 1% P/S + 1 pg/ml Puromycin. After detaching the cells with 0.05% trypsin for 5 min at 37 °C, both the “donor” and “recipient” cells were resuspended to a density of 100 million cells/ml in in PBS + 0.2% FBS. The cell suspensions were gently vortexed to mix and 10 million cells per tumor (injection volume of 100 pL) were injected subcutaneously. “Donor” tumors expressing H3-Cre were subcutaneously injected into the left flank, and “recipient” tumors harboring the “color switch” Cre-reporter were subcutaneously injected into the right flank of approximately 10-week-old female NCr mice (Taconic, #NCRNU-F) anesthetized by isoflurane inhalation. As controls H4-Cre xenografts were injected using identical methodology. 20 mice were injected per experimental group. All procedures followed approved IACUC protocols and mice were monitored daily for changes in weight, changes in activity, and food and water intake. 5 weeks later, once palpable tumors had formed, the animals were sacrificed, and the “recipient” tumors were processed for flow cytometry and immunoblot analysis as described above. The data shown represent 2 true biological replicates using different cohorts of mice and independently generated “donor” and “recipient” cells.
[00423] Mouse Fasting
[00424] For mouse fasting experiments food was removed from the cages of approximately 10-week-old Female Black 6 mice (Taconic, C57BL/6NTac) for 24 h. All procedures followed approved IACUC protocols, and the mice retained access to water, and were monitored for changes in activity and behavior. After 24 h of fasting, each mouse was put under deep isoflurane anesthesia, and up to 700 pL of whole blood was collected per animal by cardiac puncture, sacrificing the animal. Whole blood was transferred to a 1.5 mL microcentrifuge tube and incubated upright at room temperature for 30 min. The whole blood sample was then centrifuged at 1500g for 10 min at 4 °C. After centrifugation the serum was then transferred to a fresh a 1.5 mL microcentrifuge tube. Next, Albumin was depleted by incubating the serum with a solution of 5 % TCA in Acetone (1 part serum: 10 part 5% TCA in acetone) for 30 min on ice15 16. The mixture was centrifuged at 17000g for 5 min and the pelleted was saved, washed three times with 800 pL acetone followed by a 5 min centrifugation at 17,000g and 4 °C and then resuspended in 20 pL lx Sample Buffer and processed for Immunoblot as indicated.
[00425] References Cited in this Example
1 Maxwell, P. H. et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271-275 (1999). 2 Wadia, J. S., Stan, R. V. & Dowdy, S. F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nature medicine 10, 310-315 (2004).
3 Nicholson, H. E. et al. HIF -independent synthetic lethality between CDK4/6 inhibition and VHL loss across species. Science signaling 12 (2019).
4 Sulkowski, P. L. et al. Oncometabolites suppress DNA repair by disrupting local chromatin signalling. Nature 582, 586-591, doi:10.1038/s41586-020-2363-0 (2020).
5 McBrayer, S. K. et al. Transaminase inhibition by 2-hydroxyglutarate impairs glutamate biosynthesis and redox homeostasis in glioma. Cell 175, 101-116. el25 (2018).
6 Gao, W., Li, W., Xiao, T., Liu, X. S. & Kaelin, W. G. Inactivation of the PBRMl tumor suppressor gene amplifies the HIF-response in VHL-/- clear cell renal carcinoma. Proceedings of the National Academy of Sciences 114, 1027-1032 (2017).
7 Chakraborty, A. A. et al. Histone demethylase KDM6A directly senses oxygen to control chromatin and cell fate. Science 363, 1217-1222 (2019).
8 Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off- target effects of CRISPR-Cas9. Nature biotechnology 34, 184-191 (2016).
9 Katzen, F. Gateway® recombinational cloning: a biological operating system. Expert opinion on drug discovery 2, 571-589 (2007).
10 Droujinine, L A. et al. Proteomics of protein trafficking by in vivo tissue-specific labeling. Nature Communications 12, 2382, doi:10.1038/s41467-021-22599-x (2021).
11 Branon, T. C. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nature biotechnology 36, 880-887 (2018).
12 Cho, K. F. et al. Proximity labeling in mammalian cells with TurboID and split- TurboID. Nature Protocols 15, 3971-3999 (2020). 13 Serge, A., Bertaux, N., Rigneault, H. & Marguet, D. Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nature methods 5, 687-694 (2008).
14 Tarantino, N. et al. TNF and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO-IKK supramolecular structures. Journal of Cell Biology 204, 231-245 (2014).
15 Liu, G. et al. A novel and cost effective method of removing excess albumin from plasma/serum samples and its impacts on LC-MS/MS bioanalysis of therapeutic proteins. Analytical chemistry 86, 8336-8343 (2014).
16 Poltep, K., Tesena, P., Yingchutrakul, Y., Taylor, J. & Wongtawan, T. Optimisation of a serum albumin removal protocol for use in a proteomic study to identify the protein biomarkers for silent gastric ulceration in horses. Journal of equine science 29, 53-60 (2018).
EQUIVALENTS
[00426] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Claims

What is claimed:
1. An isolated or recombinant histone polypeptide comprising a peptide or fragment thereof having at least 90% identity to the amino acid sequence according to:
Figure imgf000123_0001
2. The histone polypeptide of claim 1, wherein the peptide consists of the amino acid sequence according to SEQ ID NO: 1, 2, or 3.
3. The histone polypeptide of claim 1, wherein the peptide comprises one or more modifications.
4. The histone polypeptide of claim 3, wherein the one or more modifications comprises one or more post-translational modifications and/or mutations.
5. The histone polypeptide of claim 4, wherein the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
6. The histone polypeptide of claim 4, wherein the mutation one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
7. The histone polypeptide of claim 1, wherein the fragment comprises an N-terminus truncated fragment or a C -terminus truncated fragment.
8. The histone polypeptide of claim 7, wherein the fragment comprises s fragment of SEQ ID NO: 1, 2 or 3, or a sequence at least 90% identical thereto.
9. The histone polypeptide of claim 1, wherein the histone polypeptide exhibits at least one activity selected from the group consisting of crossing a cell membrane, secretion from a cell, uptake by a cell, or delivery of a macromolecule.
10. The histone polypeptide of claim 1, wherein the histone polypeptide is a eukaryotic histone polypeptide.
11. The histone polypeptide of claim 1, wherein the histone polypeptide is a mammalian histone polypeptide.
12. The histone polypeptide of claim 1, wherein the histone polypeptide is isolated from a host cell which expresses the polypeptide.
13. The histone polypeptide of claim 12, wherein the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell.
14. The histone polypeptide of claim 1, wherein the histone polypeptide is encoded by a nucleic acid sequence according to 4, a fragment thereof, or a sequence at least 90% identical thereto.
15. The histone polypeptide of claim 1 which comprises a fusion protein.
16. The histone polypeptide of claim 15, wherein the fusion protein comprises at least one functional moiety.
17. The histone polypeptide of claim 16, wherein the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof.
18. The histone polypeptide of claim 17, wherein the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin.
19. The histone polypeptide of claim 17, wherein the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
20. The histone polypeptide of claim 16, wherein the functional moiety comprises a macromolecule.
21. The histone polypeptide of claim 20, wherein the macromolecule comprises a protein or a chemical compound.
22. The histone polypeptide of claim 21, wherein the protein comprises an antibody, a cytokine, or a growth-inhibitor.
23. The histone polypeptide of claim 21, wherein the chemical compound comprises a chemotherapeutic or toxin
24. A nucleic acid encoding a histone polypeptide or fragment thereof according to SEQ ID NO: 1, 2, or 3, or an amino acid sequence having at least 90% identity thereto.
25. The nucleic acid of claim 24, wherein the nucleic acid comprises a sequence having at least 90% identity to SEQ ID NO: 4 or a fragment thereof.
26. The nucleic acid of claim 25, wherein the nucleic acid consists of SEQ ID NO: 4.
27. The nucleic acid of claim 24, wherein the histone polypeptide or fragment thereof comprises one or more modifications.
28. The nucleic acid of claim 27, wherein the one or more modifications comprises one or more post-translational modifications and/or mutations.
29. The nucleic acid of claim 28, wherein the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
30. The nucleic acid of claim 28, wherein the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
31. A cell comprising the nucleic acid of claim 24.
32. The cell of claim 31, wherein the cell is a mammalian cell, bacterial cell, yeast cell, or an insect cell.
33. A fusion protein comprising an isolated or recombinant histone polypeptide comprising an amino acid sequence of at least 90% identity to SEQ ID NO: 1, 2, or 3 or a fragment thereof and at least one functional moiety.
34. The fusion protein of claim 33, wherein the histone polypeptide consists of SEQ ID NO: 1, 2, or 3.
35. The fusion protein of claim 33, wherein the histone polypeptide comprises one or more modifications.
36. The fusion protein of claim 35, wherein the one or more modifications comprises one or more post-translational modifications and/or mutations.
37. The fusion protein of claim 36, wherein the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
38. The fusion protein of claim 36, wherein the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
39. The fusion protein of claim 33, wherein the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof .
40. The fusion protein of claim 39, wherein the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin.
41. The fusion protein of claim 39, wherein the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
42. The fusion protein of claim 33, wherein the functional moiety comprises a macromolecule.
43. The fusion protein of claim 42, wherein the macromolecule comprises a protein or a chemical compound.
44. The fusion protein of claim 43, wherein the protein comprises an antibody, a cytokine, or a growth-inhibitor.
45. The fusion protein of claim 43, wherein the chemical compound comprises a chemotherapeutic or a toxin.
46. A cell expressing the histone polypeptide of claim 1 or the fusion protein of claim 33.
47. The cell of claim 46, wherein the cell comprises an insect cell, a mammalian cell, a bacterial, or a yeast cell.
48. A composition comprising the histone polypeptide of claim 1, the fusion protein of claim 33, or the cell of claim 46, and a pharmaceutically acceptable carrier.
49. The composition of claim 48, further comprising at least one additional active agent.
50. A drug-delivery platform comprising an isolated or recombinant histone polypeptide comprising an amino acid sequence of at least 90% identity to SEQ ID NO: 1, 2, or 3 or a fragment thereof.
51. The drug-delivery platform of claim 50, wherein the amino acid sequence of the isolate or recombinant histone polypeptide consists of an amino acid sequence according to SEQ ID NO: 1, 2 or 3.
52. The drug-delivery platform of claim 51, wherein the drug-delivery platform comprises one or more modifications.
53. The drug-delivery platform of claim 52, wherein the one or more modifications comprises one or more post-translational modifications and/or mutations.
54. The drug-delivery platform of claim 53, wherein the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
55. The drug delivery platform of claim 53, wherein the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
56. The drug-delivery platform of claim 50, comprising a fusion protein.
57. The drug-delivery platform of claim 56, wherein the fusion protein comprises at least one functional moiety.
58. The drug-delivery platform of claim 57, wherein the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof.
59. The drug-delivery platform of claim 58, wherein the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin.
60. The drug-delivery platform of claim 58, wherein the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
61. The drug-delivery platform of claim 57, wherein the functional moiety comprises a macromolecule.
62. The drug-delivery platform of claim 61, wherein the macromolecule comprises a protein or chemical compound.
63. The drug-delivery platform of claim 62, wherein the protein comprises an antibody, a cytokine, or growth-inhibitor.
64. The drug-deliver platform of claim 62, wherein the chemical compound comprises a chemotherapeutic or toxin.
65. A method of delivering at least one functional moiety to a cell, the method comprising contacting the cell with an isolated or recombinant histone polypeptide comprising the amino acid sequence of at least 90% identity to SEQ ID NO: 1, 2, or 3, or a fragment thereof, wherein the at least one functional moiety is fused to the histone polypeptide.
66. The method of claim 65 wherein the histone polypeptide consists of SEQ ID NO: 1, 2 or 3.
67. The method of claim 65, wherein the histone polypeptide comprises one or more modifications.
68. The method of claim 67, wherein the one or more modifications comprises one or more post-translational modifications and/or mutations.
69. The method of claim 68, wherein the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
70. The method of claim 68, wherein the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
71. The method of claim 65, wherein the cell is a cancer cell.
72. The method of claim 65, wherein the cell is a non-cancer cell.
73. The method of claim 65, wherein the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof.
74. The method of claim 73, wherein the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin.
75. The method of claim 73, wherein the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
76. The method of claim 65, wherein the functional moiety comprises a macromolecule.
77. The method of claim 76, wherein the macromolecule comprises a protein or chemical compound.
78. The method of claim 77, wherein the protein comprises an antibody, a cytokine, or a growth inhibitor.
79. The method of claim 77, wherein the chemical compound comprises a chemotherapeutic or toxin.
80. A method of treating a subject afflicted with a disease, the method comprising administering to the subject an isolated or recombinant histone polypeptide comprising an amino acid sequence of at least 90% identity to SEQ ID NO: 1, 2, or 3 or a fragment thereof, wherein the histone polypeptide is fused to a functional moiety.
81. The method of claim 80, wherein the histone polypeptide consists of SEQ ID NO: 1,
2, or 3.
82. The method of claim 80, wherein the histone polypeptide comprises one or more modifications.
83. The method of claim 82, wherein the one or more modifications comprises one or more post-translational modifications and/or mutations.
84. The method of claim 83, wherein the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
85. The method of claim 83, wherein the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
86. The method of claim 80, wherein the disease comprises cancer.
87. The method of claim 80, wherein the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof.
88. The method of claim 87, wherein the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin.
89. The method of claim 87, wherein the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
90. The method of claim 80, wherein the functional moiety comprises a macromolecule.
91. The method of claim 90, wherein the macromolecule comprises a protein or chemical compound.
92. The method of claim 91, wherein the protein comprises an antibody, a cytokine, or a growth inhibitor.
93. The method of claim 91, wherein the chemical compound comprises a chemotherapeutic or toxin.
94. A method of treating a subject afflicted with a disease, the method comprising administering to the subject an isolated or recombinant histone polypeptide fused to a functional moiety.
95. The method of claim 94, wherein the disease comprises cancer.
96. The method of claim 94, wherein the histone polypeptide comprises histone H3.
97. The method of claim 96, wherein the histone H3 comprises histone H3.1, histone H3.2, or histone H3.3.
98. The method of claim 96, wherein the histone polypeptide comprises an amino acid sequence or fragment thereof according to SEQ ID NO: 1, 2 or 3, or a sequence at least 90% identical thereto.
99. The method of claim 98, wherein the histone polypeptide comprises one or more modifications.
100. The method of claim 99, wherein the one or more modifications comprises one or more post-translational modifications and/or mutations.
101. The method of claim 100, wherein the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
102. The method of claim 100, wherein the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
103. The method of claim 94, wherein the functional moiety comprises a therapeutic moiety, an imaging moiety, a linker, an adjuvant, or any combination thereof.
104. The method of claim 103, wherein the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin.
105. The method of claim 103, wherein the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
106. The method of claim 94, wherein the functional moiety comprises a macromolecule.
107. The method of claim 106, wherein the macromolecule comprises a protein or chemical compound.
108. The method of claim 107, wherein the protein comprises an antibody, a cytokine, or growth inhibitor.
109. The method of claim 107, wherein the chemical compound comprises a chemotherapeutic or toxin.
110. A method of transducing a cell with a protein or chemical compound, the method comprising contacting the cell with an isolated or recombinant histone polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1, 2, or 3 or a fragment thereof, wherein the histone polypeptide is fused to the protein or chemical compound.
111. The method of claim 110, wherein the histone polypeptide consists of SEQ ID NO: 1, 2 or 3.
112. The method of claim 111, wherein the histone polypeptide comprises one or more modifications.
113. The method of claim 112, wherein the one or more modifications comprises one or more post-translational modifications and/or mutations.
114. The method of claim 113, wherein the post-translational modification comprises acetylation at Lys-14 according to SEQ ID NO: 1, 2 or 3.
115. The method of claim 113, wherein the mutation comprises one or more substitutions comprising Lys-14 to Gln-14, Lys-9 to Met-9, or Lys-27 to Met-27, according to SEQ ID NO: 1, 2 or 3.
116. The method of claim 110, wherein the cell is a cancer cell.
117. The method of claim 110, wherein the cell is a non-cancer cell.
118. The method of claim 110, wherein the protein comprises a therapeutic moiety or an imaging moiety.
119. The method of claim 118, wherein the therapeutic moiety comprises a therapeutic protein or polypeptide, a small molecule, or a toxin.
120. The method of claim 118, wherein the imaging moiety is radioactive, enzymatic, chemiluminescent, or fluorescent.
121. The method of claim 110, wherein the protein comprises an antibody, a cytokine, or growth inhibitor.
122. The method of claim 110, wherein the chemical compound comprises a chemotherapeutic or toxin.
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