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Targeting central serotonergic neurons with lentiviral vectors based on a transcriptional amplification strategy

Gene Therapy volume 16, pages 681–688 (2009)Cite this article

Abstract

Numerous pathological processes have been linked to disorders of the central 5-hydroxytryptamine (5HT) system but the possibility of gene therapy through modulation of 5HT system remained unexplored due to the lack of the specific targeting vectors. We explored the sequences upstream of the tryptophan hydroxylase-2 (TPH-2) gene, the key enzyme in neuronal 5HT synthesis and generated lentiviral vectors, which were then tested in vivo using microinjections into the rat raphe. All fragments longer than 1 kb (called 2TPH, 3.6TPH and 6.7TPH) drove highly specific expression in 5HT neurons which was, however too weak to be detectable without immunostaining. To enhance the level of expression, a two-step transcriptional amplification strategy was employed whereby the activity of a TPH-2 promoter fragment was potentiated by a chimeric enhancer GAL4∣p65. Surprisingly, previously published implementations of this strategy compromised the specificity of expression. We therefore used a new approach where both, the transgene and GAL4∣p65 are co-expressed using an internal ribosomal entry site and GAL4∣p65 operates in a positive feedback manner to amplify the expression. Thus, we have generated new vectors, which are both, highly specific for 5HT neurons and sufficiently powerful. They open a range of new opportunities for enquiries into genetic correction of 5HT-related disorders.

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Acknowledgements

KB was a recipient of the Fellowship from the Algerian Government. B-HL is funded by a British Heart Foundation Programme Grant (RG/07/006/23634). AGT was a recipient of a Fellowship from the British Heart Foundation (FS/04/003). FT is funded by the Wellcome Trust 080314/Z/06/Z.

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Authors and Affiliations

  1. Department of Physiology and Pharmacology, University of Bristol, Bristol, UK

    K Benzekhroufa, B-H Liu, A G Teschemacher & S Kasparov

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  1. K Benzekhroufa
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  2. B-H Liu
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  3. A G Teschemacher
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  4. S Kasparov
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Correspondence to S Kasparov.

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

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Benzekhroufa, K., Liu, BH., Teschemacher, A. et al. Targeting central serotonergic neurons with lentiviral vectors based on a transcriptional amplification strategy. Gene Ther 16, 681–688 (2009). https://doi.org/10.1038/gt.2009.7

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