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Improved retroviral episome transfer of transcription factors enables sustained cell fate modification

Gene Therapy volume 21, pages 938–949 (2014)Cite this article

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Abstract

Retroviral vectors are versatile gene transfer vehicles widely used in basic research and gene therapy. Mutation of retroviral integrase converts these vectors into transient, integration-deficient gene delivery vehicles associated with a high degree of biosafety. We explored the option to use integration-deficient retroviral vectors to achieve transient ectopic expression of transcription factors, which is considered an important tool for induced cell fate conversion. Stepwise optimization of the retroviral episome transfer as exemplified for the transcription factor Oct4 enabled to improve both expression magnitude and endurance. Long terminal repeat-driven γ-retroviral vectors were identified as the most suitable vector architecture. Episomal expression was enhanced by epigenetic modifiers, and Oct4 activity was increased following fusion to a minimal transactivation motif of herpes simplex virus VP16. Based on kinetic analyses, we identified optimal time intervals for repeated vector administration and established prolonged expression windows of choice. Providing proof-of-concept, episomal transfer of Oct4 was potent to mediate conversion of human fibroblasts stably expressing Klf4, Sox2 and c-Myc into induced pluripotent stem cells, which were mainly free of residual Oct4 vector integration. This study provides evidence for suitability of retroviral episome transfer of transcription factors for cell fate conversion, allowing the generation of distinct patient- or disease-specific cell types.

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Acknowledgements

This work was supported by grants from the German Academic Exchange Service (DAAD, Modern Application of Biotechnology), the Federal Ministry of Education and Research (BMBF, network projects ReGene and PidNet), the Deutsche Forschungsgemeinschaft (SPP1230, SFB738 and Cluster of Excellence REBIRTH) and the European Union (FP7 integrated projects CELL-PID and PERSIST). JWS was supported by the Hannover Biomedical Research School (HBRS; DFG, GSC 108) and the PhD program ‘Molecular Medicine’. We thank Nico Jäschke for experimental support and help with cytotoxicity assays, Michael Morgan for critical reading of the manuscript, Malte Sgodda for H9 hESC RNA (all Hannover Medical School, Hannover, Germany), Gerald Draeger for providing ROCK inhibitor Y-27632 and Thomas Scheper for basic fibroblast growth factor (both Leibniz University Hannover, Hannover, Germany).

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Authors and Affiliations

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

    J W Schott, D Hoffmann, T Maetzig, D Zychlinski, C Baum & A Schambach

  2. Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany

    J W Schott, D Hoffmann, T Maetzig, D Steinemann, D Zychlinski, T Cantz, C Baum & A Schambach

  3. Center for Integrated Psychiatry, Christian Albrechts University, Kiel School of Medicine, Kiel, Germany

    F-J Müller

  4. Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany

    D Steinemann

  5. Translational Hepatology and Stem Cell Biology, Hannover Medical School, Hannover, Germany

    T Cantz

  6. Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA,

    A Schambach

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  1. J W Schott
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Correspondence to A Schambach.

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Schott, J., Hoffmann, D., Maetzig, T. et al. Improved retroviral episome transfer of transcription factors enables sustained cell fate modification. Gene Ther 21, 938–949 (2014). https://doi.org/10.1038/gt.2014.69

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