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Epidermal growth factor improves lentivirus vector gene transfer into primary mouse hepatocytes

Gene Therapy volume 19, pages 425–434 (2012)Cite this article

Abstract

Partial resistance of primary mouse hepatocytes to lentiviral (LV) vector transduction poses a challenge for ex vivo gene therapy protocols in models of monogenetic liver disease. We thus sought to optimize ex vivo LV gene transfer while preserving the hepatocyte integrity for subsequent transplantation into recipient animals. We found that culture media supplemented with epidermal growth factor (EGF) and, to a lesser extent, hepatocyte growth factor (HGF) markedly improved transduction efficacy at various multiplicities of infection. Up to 87% of primary hepatocytes were transduced in the presence of 10 ng EGF, compared with ∼30% in standard culture medium (SCMs). The increased number of transgene-expressing cells correlated with increased nuclear import and more integrated pro-viral copies per cell. Higher LV transduction efficacy was not associated with proliferation, as transduction capacity of gammaretroviral vectors remained low (<1%). Finally, we developed an LV transduction protocol for short-term (maximum 24 h) adherent hepatocyte cultures. LV-transduced hepatocytes showed liver repopulation capacities similar to freshly isolated hepatocytes in alb-uPA mouse recipients. Our findings highlight the importance of EGF for efficient LV transduction of primary hepatocytes in culture and should facilitate studies of LV gene transfer in mouse models of monogenetic liver disease.

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Acknowledgements

We thank Sabine Knöß and Johanna Krause for excellent technical help, and Qinggong Yuan for help with transplantation of cells (all from Hannover Medical School, Hannover, Germany). The LV ID gag/pol construct (pcDNA3.GP.4 × CTE with D64V mutation) was provided by M Milsom (Boston, MA, USA, and DKFZ, Heidelberg, Germany). The study was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 738, SPP 1230 and the Excellence Cluster REBIRTH). The work was supported by the SFB 738 ‘Improvement of conventional and innovative transplants’ (MO, UM), the Cluster of Excellence ‘Rebirth’ (MO) and the Grand Challenge Program of the Bill and Melinda Gates Foundation (MO).

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Author notes

  1. M Rothe and I Rittelmeyer: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany

    M Rothe, I Rittelmeyer, M Iken, U Rüdrich, M P Manns, M Bock & M Ott

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

    M Rothe, A Schambach, C Baum & U Modlich

  3. TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Hannover Medical School (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany

    M Rothe, I Rittelmeyer, M Iken, U Rüdrich & M Ott

  4. Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany

    S Glage

  5. Junior Research Group Hepatic Cell Therapy, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany

    M Bock

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  1. M Rothe
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Correspondence to M Ott or U Modlich.

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Rothe, M., Rittelmeyer, I., Iken, M. et al. Epidermal growth factor improves lentivirus vector gene transfer into primary mouse hepatocytes. Gene Ther 19, 425–434 (2012). https://doi.org/10.1038/gt.2011.117

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