Anesti, A.-M.; (2009) Development of replication-defective herpes simplex viral vectors for delivery of RNA interference to neurons of the peripheral nervous system. Doctoral thesis , UCL (University College London).

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Abstract

Considerable interest has been focused on inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although small interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi to the central and peripheral nervous system remains a major challenge limiting its applications. This thesis describes the development of a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) using replication-defective herpes simplex viral (HSV-1) vectors by identifying and evaluating various approaches to induce RNAi, i.e. expression of individual short-hairpin RNAs (shRNAs), artificial microRNAs (miRNAs) and multiple tandem miRNAs. Following the development of these systems, HSV-mediated delivery of shRNA or miRNA against reporter genes was shown to result in highly effective and specific silencing in neuronal and non-neuronal cells in culture and in the DRG of mice in vivo, including in a transgenic mouse model. Proof of concept was established by demonstrating in vivo silencing of the endogenous trpv1 gene, thought to be involved in nociception, by assessing both mRNA and protein levels. These data are the first to show silencing in DRG neurons in vivo by vector-mediated delivery of shRNA and support the utility of HSV vectors for gene silencing in peripheral neurons and the potential application of this technology to the study of nociceptive processes and in pain gene target validation studies. Moreover, a disabled HSV-1 vector targeting p75, Lingo1 and NgR2, which are involved in myelin inhibition of axonal regeneration, was developed and evaluated for its ability to promote regeneration of sensory axons into the spinal cord, following injury of the dorsal roots. This is the first time such an appoach to silencing multiple genes has been employed. Although HSV-mediated delivery of multiple miRNAs resulted in highly effective silencing of these genes in dividing cells in culture, while highly effective silencing of p75 was achieved, only modest silencing of Lingo1 and NgR2 was observed in DRG neurons in vivo. Preliminary regeneration experiments, which were largely outside the scope of this thesis, were inconclusive and require more extensive study as a stand-alone project, if the in vivo potential of the approach developed for silencing multiple genes targeted at axonal regeneration is to be further explored.

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