title: Tightly regulated 'all-in-one' lentiviral vectors for protection of human hematopoietic cells from anticancer chemotherapy.
abstract: Successful application of gene therapy strategies may require stringently regulated transgene expression. Along this line, we describe a doxycycline (Dox)-inducible 'all-in-one' lentiviral vector design using the pTET-T11 (TII) minimal-promoter and a reverse transactivator protein (rtTA2S-M2) driven by the phosphoglycerate kinase promoter allowing for tight regulation of transgene expression (Lv.TII vectors). Vector design was evaluated in human hematopoietic cells in the context of cytidine deaminase (hCDD)-based myeloprotective gene therapy. Upon Dox administration, a rapid (16-24 h) and dose-dependent (>0.04 μg ml(-1) Dox) onset of transgene expression was detected in Lv.TII.CDD gene-modified K562 cells as well as in primary human CD34(+) hematopoietic cells. Importantly, in both cell models low background transgene expression was observed in the absence of Dox. Functionality of Dox-inducible hCDD expression was demonstrated by >10-fold increase in cytosine arabinoside (1-β-d-arabinofuranosylcytosine, Ara-C) resistance of Lv.TII.CDD-transduced K562 cells. In addition, Lv.TII.CDD-transduced CD34(+)-derived myeloid cells were protected from up to 300 nm Ara-C (control affected from 50 nm onwards). These data clearly demonstrate the suitability of our self-inactivating lentiviral vector to induce robust, tightly regulated transgene expression in human hematopoietic cells with minimal background activity and highlight the potential of our construct in myeloprotective gene therapy strategies.
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Lachmann
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Brennig
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Hillje
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Schermeier
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Phaltane
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Dahlmann
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany.
authors.forename: I
authors.initials: I
authors.lastname: Gruh
authors.affiliation: LOEWE-Research Group for (targeted) Gene Modification in Stem Cells, Paul-Ehrlich-Institute, Langen, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Heinz
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Schiedlmeier
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Baum
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Moritz
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000633:toxicity; D003561:Cytarabine / Q000633:toxicity; D003564:Cytidine Deaminase / Q000096:biosynthesis / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000187:drug effects* / Q000502:physiology* / Q000821:virology; D006801:Humans; D020014:K562 Cells; D016086:Lentivirus / Q000235:genetics*; D061251:Primary Cell Culture; D011401:Promoter Regions, Genetic; D019076:Transgenes
chemical_list: D000964:Antimetabolites, Antineoplastic; D003561:Cytarabine; D003564:Cytidine Deaminase; D004318:Doxycycline