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Gene Ther ; 22(11): 883-92, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26125609

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-ß-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.


Subject(s)
Genetic Therapy/methods , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/physiology , Lentivirus/genetics , Antimetabolites, Antineoplastic/toxicity , Cytarabine/toxicity , Cytidine Deaminase/biosynthesis , Cytidine Deaminase/genetics , Doxycycline/pharmacology , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Hematopoietic Stem Cells/virology , Humans , K562 Cells , Primary Cell Culture , Promoter Regions, Genetic , Transgenes
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