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Influence of multiplicity of infection and protein stability on retroviral vector-mediated gene expression in hematopoietic cells

Gene Therapy volume 8, pages 477–486 (2001)Cite this article

Abstract

Using retroviral vectors encoding enhanced green fluorescent protein (egfp), we addressed to what extent expression of retroviral transgenes in hematopoietic cells depends on the multiplicity of infection (moi) and on the half-life of the encoded protein. we show that an elevation of the moi not only elevates the frequency of transduced cells, but also increases transgene expression levels and reduces interanimal variability in vivo (hematopoietic cells of c57bl/6j mice analyzed 13 weeks after transplantation). this suggests that the moi has to be carefully controlled and should be adapted as desired for clinical studies when evaluating vector performance in preclinical models. the impact of protein stability is demonstrated by comparing vectors expressing egfp or a destabilized variant with a c-terminal pest-sequence, d2egfp. the loss of expression with d2egfp was more pronounced in terminally differentiated cells of the peripheral blood (>30 fold) than in progenitor cells (five- to 10-fold), indicating a stronger transcription of the retroviral promoter in progenitor cells and a predominant role of protein inheritance over de novo synthesis of transgenic protein in mature blood cells. This analysis reveals an important and differentiation-dependent contribution of protein half-life to the expression of retroviral vectors in hematopoietic cells, establishes d2EGFP as a more accurate reporter for determination of vector transcription, and also suggests that preclinical data obtained under conditions of high transduction rates or with vectors expressing stable reporter proteins require careful interpretation.

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Acknowledgements

We thank Cordula Grüttner for excellent technical assistance. This work contains parts of the doctoral thesis of Anke Wahlers (University of Hamburg, Faculty of Biology). This work was supported by the Deutsche Krebshilfe (10–1456-Ba2), by the Erich and Gertrud Roggenbuck-Stiftung and by the Hamburger Stiftung zur Foerderung der Krebsbekämpfung. The Heinrich-Pette-Institute is financially supported by the Freie und Hansestadt Hamburg and by the Bundesministerium für Gesundheit.

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Author notes

  1. C Baum

    Present address: Laboratory for Experimental Cell Therapy, Dept. Hematology and Oncology, Hannover Medical School, Carl-Neuberg-Str. 1, Hannover, 30625, Germany

Authors and Affiliations

  1. Department Cell and Virus Genetics, Heinrich-Pette-Institute, Hamburg, Germany

    A Wahlers, M Schwieger, Z Li, D Meier-Tackmann, D von Laer & C Baum

  2. EUFETS GmbH, Idar-Oberstein, Hamburg, Germany

    C Lindemann & H-G Eckert

  3. Bone Marrow Transplantation, University Hospital Eppendorf, Hamburg, Germany

    C Baum

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  1. A Wahlers
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Wahlers, A., Schwieger, M., Li, Z. et al. Influence of multiplicity of infection and protein stability on retroviral vector-mediated gene expression in hematopoietic cells. Gene Ther 8, 477–486 (2001). https://doi.org/10.1038/sj.gt.3301426

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