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Rapid identification of novel functional promoters for gene therapy

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Abstract

Transcriptional control of transgene expression is crucial to successful gene therapy, yet few promoter/enhancer combinations have been tested in clinical trials. We created a simple, desktop computer database and populated it with promoter sequences from publicly available sources. From this database, we rapidly identified novel CpG-free promoter sequences suitable for use in non-inflammatory, non-viral in vivo gene transfer. In a simple model of lung gene transfer, five of the six promoter elements selected, chosen without prior knowledge of their transcriptional activities, directed significant transgene expression. Each of the five novel promoters directed transgene expression for at least 14 days post-delivery, greatly exceeding the duration achieved with the commonly used CpG-rich viral enhancer/promoters. Novel promoter activity was also evaluated in a more clinically relevant model of aerosol-mediated lung gene transfer and in the liver following delivery via high-pressure tail vein injection. In each case, the novel CpG-free promoters exhibited higher and/or more sustained transgene expression than commonly used CpG-rich enhancer/promoter sequences. This study demonstrates that novel CpG-free promoters can be readily identified and that they can direct significant levels of transgene expression. Furthermore, the database search criteria can be quickly adjusted to identify other novel promoter elements for a variety of transgene expression applications.

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Acknowledgments

The work was supported by a grant from the Cystic Fibrosis Trust to the UK Cystic Fibrosis Gene Therapy Consortium.

Conflict of interest

The authors declare no conflicts of interest.

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Authors and Affiliations

  1. GeneMedicine Research Group, NDCLS, John Radcliffe Hospital, University of Oxford, OX3 9DU, Oxford, UK

    Ian A. Pringle, Deborah R. Gill, Mary M. Connolly, Anna E. Lawton, Anne-Marie Hewitt, Graciela Nunez-Alonso, Lee A. Davies & Stephen C. Hyde

  2. Genzyme Corporation, Framingham, MA, USA

    Seng H. Cheng & Ronald K. Scheule

  3. The UK Cystic Fibrosis Gene Therapy Consortium, Oxford, UK

    Ian A. Pringle, Deborah R. Gill, Mary M. Connolly, Anna E. Lawton, Anne-Marie Hewitt, Graciela Nunez-Alonso, Lee A. Davies & Stephen C. Hyde

Authors
  1. Ian A. Pringle
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  2. Deborah R. Gill
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  3. Mary M. Connolly
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  4. Anna E. Lawton
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  5. Anne-Marie Hewitt
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  6. Graciela Nunez-Alonso
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  7. Seng H. Cheng
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  8. Ronald K. Scheule
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  9. Lee A. Davies
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  10. Stephen C. Hyde
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Corresponding author

Correspondence to Stephen C. Hyde.

Additional information

Ian A. Pringle and Deborah R. Gill contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 953 kb)

ESM 1

The FileMaker Pro version of the PromoSer database contains 62571 promoter sequences. (ZIP 51.5 MB)

ESM 1

The FileMaker Pro version of the EPD database contains ~4000 promoter sequences. (ZIP 4.20 MB)

ESM 1

The FileMaker Pro databases have the facility to count the total number of binding sites for a user-defined 'family' of (up to 100) arbitrary DNA sequences (eg transcription factor binding sites). Instructions for how to perform this are included in the databases. (ZIP 13.1 kb)

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Pringle, I.A., Gill, D.R., Connolly, M.M. et al. Rapid identification of novel functional promoters for gene therapy. J Mol Med 90, 1487–1496 (2012). https://doi.org/10.1007/s00109-012-0928-6

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  • DOI: https://doi.org/10.1007/s00109-012-0928-6

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