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Lentiviral vectors with amplified β cell-specific gene expression

Gene Therapy volume 16, pages 998–1008 (2009)Cite this article

Abstract

An important goal of gene therapy is to be able to deliver genes, so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Although tissue-specific promoters confer selectivity, in a vector-based system, their activity may be too weak to mediate detectable levels in gene-expression studies. We have used a two-step transcriptional amplification system to amplify gene expression from lentiviral vectors using the human insulin promoter. In this system, the human insulin promoter drives expression of a potent synthetic transcription activator (the yeast GAL4 DNA-binding domain fused to the activation domain of the Herpes simplex virus-1 VP16 activator), which in turn activates a GAL4-responsive promoter, driving the enhanced green fluorescent protein reporter gene. Vectors carrying the human insulin promoter did not express in non-β-cell lines, but expressed in murine insulinoma cell lines, indicating that the human insulin promoter was capable of conferring cell specificity of expression. The insulin-amplifiable vector was able to amplify gene expression five to nine times over a standard insulin-promoter vector. In primary human islets, gene expression from the insulin-promoted vectors was coincident with insulin staining. These vectors will be useful in gene-expression studies that require a detectable signal and tissue specificity.

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Acknowledgements

We thank Denise A Carbonaro-Sarracino for technical assistance with Taqman assays, Ewa Zielinska and Lora Barsky for technical assistance with cell sorting, Karen A Pepper for assistance with molecular assays, and Xingchao Wang for technical assistance with immunohistochemistry assays. We also thank the Cellular Image Core of Childrens Hospital Los Angeles. We acknowledge the use of tissues procured by Islet Cell Resource Centers Basic Science Islet Distribution Program and the National Disease Research Interchange with support from the National Institutes of Health grant 5 U42 RR006042-17. Additional support was provided by awards from the National Institutes of Health: 5F32 AI056894 (KLS), 1R21 DK62649 (DBK), 1RO1 DK68719 (GMC), and from the Juvenile Diabetes Research Foundation: 17-2006-1137 (DBK).

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Authors and Affiliations

  1. Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, Los Angeles, CA, USA

    K L Shaw, E Pais, S Ge, C Hardee, D Skelton, R P Hollis, G M Crooks & D B Kohn

  2. Department of Pediatrics, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

    G M Crooks & D B Kohn

  3. Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

    D B Kohn

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  1. K L Shaw
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Correspondence to D B Kohn.

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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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Shaw, K., Pais, E., Ge, S. et al. Lentiviral vectors with amplified β cell-specific gene expression. Gene Ther 16, 998–1008 (2009). https://doi.org/10.1038/gt.2009.49

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