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Tightly regulated ‘all-in-one’ lentiviral vectors for protection of human hematopoietic cells from anticancer chemotherapy

Gene Therapy volume 22, pages 883–892 (2015)Cite this article

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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.

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Acknowledgements

We thank Matthias Ballmaier (PhD) and his team from the Core Facility Cell Sorting of Hannover Medical School for cell sorting and Doreen Lüttge (Hannover Medical School) for excellent technical assistance. Furthermore, we thank Georg Kensah (Hannover Medical School, now Otto-von-Guericke University, Magdeburg, Germany) for help in performing the time-lapse video studies. This work was supported by grants from the Deutsche Forschungsgemeinschaft: Cluster of Excellence REBIRTH (Exc 62/1), SPP1230 Grant MO 886/3-1 (to TM), Grant MO 886/4-1 (TM), the EU framework program grant PERSIST (to CHB) and Hannover Biomedical Research School (HBRS; DFG, GSC 108).

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Author notes

  1. N Lachmann and S Brennig: These authors contributed equally to this work.

  2. J Dahlmann: Current adress: Department of Cardiothoracic Surgery (KCHH), Otto-von-Guericke University, Magdeburg, Germany.

Authors and Affiliations

  1. Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany

    N Lachmann, S Brennig, R Hillje, H Schermeier, R Phaltane & T Moritz

  2. Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany

    N Lachmann, S Brennig, R Hillje, H Schermeier, R Phaltane, B Schiedlmeier, C Baum & T Moritz

  3. Department of Cardiothoracic, Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany

    J Dahlmann & I Gruh

  4. LOEWE-Research Group for (targeted) Gene Modification in Stem Cells, Paul-Ehrlich-Institute, Langen, Germany

    N Heinz

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Lachmann, N., Brennig, S., Hillje, R. et al. Tightly regulated ‘all-in-one’ lentiviral vectors for protection of human hematopoietic cells from anticancer chemotherapy. Gene Ther 22, 883–892 (2015). https://doi.org/10.1038/gt.2015.61

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