title: Development of an inducible platform for intercellular protein delivery.
abstract: A challenge to protein based therapies is the ability to produce biologically active proteins and their ensured delivery. Various approaches have been utilised including fusion of protein transduction domains with a protein or biomolecule of interest. A compounding issue is lack of specificity, efficiency and indeed whether the protein fusions are actually translocated into the cell and not merely an artefact of the fixation process. Here we present a novel platform, allowing the inducible export and uptake of a protein of interest. The system utilises a combination of the Tetracyline repressor system, combined with a fusion protein containing the N-terminal signal peptide from human chorionic gonadotropin beta-subunit, and a C-terminal poly-arginine domain for efficient uptake by target cells. This novel platform was validated using enhanced green fluorescent protein as the gene of interest. Doxycycline efficiently induced expression of the fusion protein. The human chorionic gonadotropin beta-subunit facilitated the export of the fusion protein into the cell culture media. Finally, the fusion protein was able to efficiently enter into neighbouring cells (target cells), mediated by the poly-arginine cell penetrating peptide. Importantly we have addressed the issue of whether the observed uptake is an artefact of the fixation process or indeed genuine translocation. In addition this platform provides a number of potential applications in diverse areas such as stem cell biology, immune therapy and cancer targeting therapies.
authors.affiliation: Department of Molecular Medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, PO Box 1112 Blindern, Oslo 0317, Norway; Norwegian Center for Stem Cell Research, PO Box 1112 Blindern, Oslo 0317, Norway.
authors.forename: Richard
authors.initials: R
authors.lastname: Siller
authors.affiliation: Tampere University, IMT, Tampere Fin-33014, Finland.
authors.forename: Eric
authors.initials: E
authors.lastname: Dufour
authors.affiliation: Department of Molecular Medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, PO Box 1112 Blindern, Oslo 0317, Norway.
authors.forename: Max
authors.initials: M
authors.lastname: Lycke
authors.affiliation: MRC Centre for Regenerative Medicine, SCRM Building, University of Edinburgh, Bioquarter, 5 Little France Drive, Edinburgh EH16 4UU, UK.
authors.forename: Ian
authors.initials: I
authors.lastname: Wilmut
authors.affiliation: Stem Cell Center, Yale, 10 Amistad 201B, New Haven, CT 06520, USA.
authors.forename: Yong-Wook
authors.initials: YW
authors.lastname: Jung
authors.affiliation: Stem Cell Center, Yale, 10 Amistad 201B, New Haven, CT 06520, USA.
authors.forename: In Hyun
authors.initials: IH
authors.lastname: Park
authors.affiliation: Department of Molecular Medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, PO Box 1112 Blindern, Oslo 0317, Norway; Norwegian Center for Stem Cell Research, PO Box 1112 Blindern, Oslo 0317, Norway; Institute of Immunology, Oslo University Hospital-Rikshospitalet, PO Box 4950 Nydalen, Oslo 0424, Norway. Electronic address: gareth.sullivan@medisin.uio.no.
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Sullivan
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002477:Cells / Q000378:metabolism; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000008:administration & dosage / Q000493:pharmacokinetics; D004318:Doxycycline / Q000494:pharmacology; D016503:Drug Delivery Systems / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D016685:Mitomycin / Q000494:pharmacology; D010455:Peptides / Q000008:administration & dosage / Q000493:pharmacokinetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000008:administration & dosage*; D011993:Recombinant Fusion Proteins; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D018997:Chorionic Gonadotropin, beta Subunit, Human; D010455:Peptides; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C015462:polyarginine; D016685:Mitomycin; D004318:Doxycycline
keywords: Cell penetrating peptides; Cell therapy; Cellular reprogramming; Gene therapy; Poly-arginine; Protein delivery; Stem cells