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

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.

Cardioprotection evoked by remote ischaemic preconditioning is critically dependent on the activity of vagal pre-ganglionic neurones

“…In this study, ChIEFtdTomato was engineered into an AV under the control of an enhanced 17,18 PRSx8 promoter. 10,11 AVs were produced as described previously.…”

Serotonin: a regulator of neuronal morphology and circuitry

“…Engineered transgenes and fluorescent reporters under control of the serotonin-specific Pet-1 enhancer region are available in mice [94] and zebrafish [95] and this technique has allowed the investigation of developmental serotonergic neuronal migration in slice culture [18] and development of the 5-HT 1A conditional knockout that is selectively targeted to serotonergic neurons [62]. In rats, viral vectors have been delivered to central serotonergic neurons in vivo and in slice culture, allowing expression of fluorescent reporters and visualization of serotonergic projections [96,97]. Recent identification of discrete transcriptional gene programming in subpopulations of raphe serotonergic neurons [98] may provide new genetic tools, and may reveal differential mechanisms responsible for functional diversity [99] and the disparate susceptibility of these neurons to toxins such as MDMA and fenfluramine [84].…”

Optogenetics in the behaving rat: integration of diverse new technologies in a vital animal model

“…Although most such genetic control regions are too large to package into AAV or LV, cell-subtype specific opsin expression has been successfully demonstrated in (for example) rat serotonin [59], somatostatin [60], CaMKIIα [61], and GFAP [25]-expressing cells using this method [28]. An important cautionary note is that a promoter fragment showing specificity in one species or in one virus type may not show the same specificity in another preparation.…”