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Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels

Gene Therapy volume 18, pages 501–508 (2011)Cite this article

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Abstract

The success of gene therapy hinges on achievement of adequate transgene expression. To ensure high transgene expression, many gene-therapy vectors include highly active virus-derived transcriptional elements. Other vectors include tissue-specific eukaryotic transcriptional elements, intended to limit transgene expression to specific cell types, avoid toxicity and prevent immune responses. Unfortunately, tissue specificity is often accompanied by lower transgene expression. Here, we use eukaryotic (murine) transcriptional elements and a virus-derived posttranscriptional element to build cassettes designed to express a potentially therapeutic gene (interleukin (IL)-10) in large-vessel endothelial cells (ECs) at levels as high as obtained with the cytomegalovirus (CMV) immediate early promoter, while retaining EC specificity. The cassettes were tested by incorporation into helper-dependent adenoviral vectors, and transduction into bovine aortic EC in vitro and rabbit carotid EC in vivo. The murine endothelin-1 promoter showed EC specificity, but expressed only 3% as much IL-10 mRNA as CMV. Inclusion of precisely four copies of an EC-specific enhancer and a posttranscriptional regulatory element increased IL-10 expression to a level at or above the CMV promoter in vivo, while retaining—and possibly enhancing—EC specificity, as measured in vitro. The cassette reported here will likely be useful for maximizing transgene expression in large-vessel EC, while minimizing systemic effects.

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Acknowledgements

We thank AdVec, Inc. for permission to use the HDAd reagents, David Russell for providing the WPRE, Axel Schambach for the oPRE sequence, Harvey Perkins for the rabbit IL-10 cDNA, Steve Hauschka for helpful discussions, and Thomas Quertermous for the murine ET-1 sequences. Sofia Penev provided valuable technical help, and we are grateful to Margo Weiss for administrative assistance. This work was supported by National Institutes of Health grant HL076226 and the John L Locke, Jr Charitable Trust. Jordan Kho was supported in part by a grant to the University of Washington from the Howard Hughes Medical Institute through the Undergraduate Science Education Program. This work has been funded by the NIH.

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  1. Department of Medicine, University of Washington, Seattle, WA, USA

    N Dronadula, L Du, R Flynn, J Buckler, J Kho, Z Jiang, S Tanaka & D A Dichek

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  1. N Dronadula
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Correspondence to D A Dichek.

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Dronadula, N., Du, L., Flynn, R. et al. Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels. Gene Ther 18, 501–508 (2011). https://doi.org/10.1038/gt.2010.173

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