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Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors

Gene Therapy volume 9, pages 477–487 (2002)Cite this article

Abstract

Oncoretroviral vectors have been successfully used in gene therapy trials, yet low transduction rates and loss of transgene expression are still major obstacles for their application. To overcome these problems we modified the widely used Moloney murine leukemia virus-derived retroviral vector pMX by replacing the 3′LTR with the spleen focus-forming virus LTR and inserting the woodchuck hepatitis B virus post-translational regulatory element. To compare requirements crucial for efficient transgene expression, we generated the hybrid retroviral vectors pMOWS and pOWS that harbor the complete murine embryonic stem cell virus (MESV)-leader sequence or a shortened MESV-leader not comprising primer binding site (PBS) and splice donor (SD). Applying these retroviral vectors significantly augmented transgene expression in hematopoietic cell lines and progenitor cells. For transduction of murine embryonic stem (ES) cells the retroviral vector pMOWS that harbors the MESV-PBS and -SD was superior resulting in 65% green fluorescent protein (GFP) expressing ES cells. Surprisingly, in murine and human primitive hematopoietic progenitor cells (HPC), the highest efficiency of up to 66% GFP expressing cells was achieved with pOWS, a retroviral vector that retains the negative regulatory element coinciding with the MoMuLV-PBS. In summary our hybrid retroviral vectors facilitate significantly improved transgene expression in multipotent cells and thus possess great potential for reconstituting genes in primary cells of disease models, as well as for gene therapy.

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Acknowledgements

We thank Dr Christopher Baum (Heinrich-Pette-Institut für Experimentelle Virology und Immunologie, Universität Hamburg) for providing the retroviral vector pSFβ1, Dr Michael Nassal (Department of Internal Medicine II, University Hospital Freiburg) for supplying the woodchuck hepatitis virus vector pCWT, and Dr Albrecht Müller for providing antibodies against Ter119 and Gr1 and helpful advice with murine hematopoietic cell cultures. We are indebted to Dr Ira Swameye for helpful discussion, Melanie Wickert for technical assistance, and Dr Frank Reuss and Dr Randy Cassada for critically reading the manuscript. This work was supported by Sonderforschungsbereich 364 (Deutsche Forschungsgemeinschaft).

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Authors and Affiliations

  1. Hans-Spemann Laboratories, Max-Planck Institute of Immunobiology, Freiburg, Germany

    R Ketteler, O Sandra & U Klingmüller

  2. Department of Hematology and Oncology, Medical University Center Freiburg, Freiburg, Germany

    S Glaser & U M Martens

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  1. R Ketteler
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Ketteler, R., Glaser, S., Sandra, O. et al. Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors. Gene Ther 9, 477–487 (2002). https://doi.org/10.1038/sj.gt.3301653

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