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Detailed comparison of retroviral vectors and promoter configurations for stable and high transgene expression in human induced pluripotent stem cells

Gene Therapy volume 24, pages 298–307 (2017)Cite this article

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Abstract

Correction of patient-specific induced pluripotent stem cells (iPSC) upon gene delivery through retroviral vectors offers new treatment perspectives for monogenetic diseases. Gene-modified iPSC clones can be screened for safe integration sites and differentiated into transplantable cells of interest. However, the current bottleneck is epigenetic vector silencing. In order to identify the most suitable retroviral expression system in iPSC, we systematically compared vectors from different retroviral genera, different promoters and their combination with ubiquitous chromatin opening elements (UCOE), and several envelope pseudotypes. Lentiviral vectors (LV) pseudotyped with vesicular stomatitis virus glycoprotein were superior to gammaretroviral and alpharetroviral vectors and other envelopes tested. The elongation factor 1α short (EFS) promoter mediated the most robust expression, whereas expression levels were lower from the potent but more silencing-prone spleen focus forming virus (SFFV) promoter. Both full-length (A2UCOE) and minimal (CBX3) UCOE juxtaposed to two physiological and one viral promoter reduced transgene silencing with equal efficiency. However, a promoter-specific decline in expression levels was not entirely prevented. Upon differentiation of transgene-positive iPSC into endothelial cells, A2UCOE.EFS and CBX3.EFS vectors maintained highest transgene expression in a larger fraction of cells as compared with all other constructs tested here. The function of UCOE diminished, but did not fully counteract, vector silencing and possibilities for improvements remain. Nevertheless, the CBX3.EFS in a LV background exhibited the most promising promoter and vector configuration for both high titer production and long-term genetic modification of human iPSC and their progeny.

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Acknowledgements

This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB738, Cluster of Excellence REBIRTH (EXC 62/1) and Mo886/6-1), the Bundesministerium für Bildung und Forschung (BMBF, Joint Research Project IFB-Tx, pid.net), the Deutscher Akademischer Austauschdienst (DAAD) and the European Union (FP7 projects PERSIST and CELLPID). FJM was supported by grants from the BMBF (13GW0128A and 01GM1513D) and from the Deutsche Forschungsgemeinschaft (German Research Foundation DFG MU 3231/3-1). We thank Johannes Kühle for providing viral supernatants, Gerald Draeger and Thomas Scheper for providing Rock inhibitor Y-27632, SB431542 inhibitor and bFGF (Leibnitz University, Hannover, Germany) as well as Malte Sgodda and Tobias Cantz for H9 ESC RNA (Hannover Medical School, Hannover, Germany). We would also like to thank Manuel Grez for his kind advice and support of this project and Adrian Schwarzer for his advice in statistical analyses.

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Authors and Affiliations

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

    D Hoffmann, J W Schott, F K Geis, L Lange, D Lenz, D Zychlinski, M Morgan, T Moritz & A Schambach

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

    D Hoffmann, J W Schott, F K Geis, L Lange, T Moritz & A Schambach

  3. Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Hannover, Germany

    D Hoffmann & A Schambach

  4. Zentrum für Integrative Psychiatrie, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany

    F-J Müller

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

    D Steinemann

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

    A Schambach

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  1. D Hoffmann
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Hoffmann, D., Schott, J., Geis, F. et al. Detailed comparison of retroviral vectors and promoter configurations for stable and high transgene expression in human induced pluripotent stem cells. Gene Ther 24, 298–307 (2017). https://doi.org/10.1038/gt.2017.20

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