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Selectable bicistronic vectors in skin gene therapy

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Abstract

Bicistronic vectors (BCV) are important tools for gene therapy applications allowing selection for increased expression of a desired gene by linking it to a selectable gene, such as the multi-drug resistance (MDR) gene. However, both the design of the BCV and the cell type to be transduced can have a strong impact on the vector performance in the target cells. To analyze which factors might influence the efficiency of BCV in achieving high gene expression levels in skin and to determine the best suited BCV for cutaneous transduction, both keratinocytes (KC) and fibroblasts (FB) were transduced with different BCV constructs, BGIM, BMIG and QGIM. In BGIM, expression of the BCV cassette encompassing the green fluorescent protein (GFP) gene connected to the MDR gene was driven by a retroviral LTR-promoter. In BMIG, the order of the two genes was reversed, while in QGIM the GFP- and MDR-gene were arranged similar as in BGIM, but expressed by a CMV- instead of an LTR-promoter. FACS-analysis revealed that the percentage of genetically modified cells varied substantially with 47.9% QGIM-, 35.5% BMIG- and 17.9% BGIM-transduced KC expressing both genes. For FB the numbers were 56.7% (QGIM), 38.4% (BMIG) and 8.3% (BGIM). Furthermore, the choice of BCV determined the intensity of GFP-expression with the highest levels measured in BGIM-, followed by QGIM- and then BMIG-transduced cells. Interestingly, highly efficient enrichment through colchicine selection was possible for QGIM- (up to 97.1% KC, 97.8% FB) and BMIG- (85.0% KC and 98.0% FB) but not BGIM- (29.9% KC and 18.6% FB) transduced cells. Finally, immunohistochemistry and FACS-analysis demonstrated, that colchicine selection of QGIM-transduced skin equivalents led to increased numbers of GFP-expressing KC (from 51.2% up to 72.3%) and enhanced GFP-intensity in the skin. These results show that BCV present a promising vector system to enhance the expression of a desired gene in skin but important parameters taken into account when employing a selectable BCV for skin gene therapy applications are the retroviral vector backbone, the order in which the genes are arranged, and the target cells to be transduced and selected.

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Abbreviations

BCV:

Bicistronic vector

ECMV:

Encephalomyocarditis virus

FB:

Fibroblasts

GFP:

Green fluorescent protein

IRES:

Internal ribosome entry site

KC:

Keratinocytes

MDR:

Multi-drug resistance

SE:

Skin equivalent

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Acknowledgements

This work was supported by Grant No. Pf 344/2-2. Jean-Philippe Therrien is a recipient of a postdoctoral fellowship from the Canadian Institutes of Health Research. The authors thank Ines Nachtigall for expert technical assistance.

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Authors and Affiliations

  1. Department of Dermatology and Allergology, University of Munich, Munich, Germany

    Frank Scheidemann & Wolfgang Pfützner

  2. Dermatology Branch, NCI, National Institutes of Health, Bethesda, MD, USA

    Jean-Philippe Therrien

  3. Department of Dermatology and Allergology, University of Giessen and Marburg, Deutschhausstr. 9, 35037, Marburg, Germany

    Wolfgang Pfützner

Authors
  1. Frank Scheidemann
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  2. Jean-Philippe Therrien
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  3. Wolfgang Pfützner
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Correspondence to Wolfgang Pfützner.

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Scheidemann, F., Therrien, JP. & Pfützner, W. Selectable bicistronic vectors in skin gene therapy. Arch Dermatol Res 300, 415–423 (2008). https://doi.org/10.1007/s00403-008-0878-6

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  • DOI: https://doi.org/10.1007/s00403-008-0878-6

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