Optimisation of a novel molecular sensor for central nervous system gene therapy

Abstract

Gene therapy is a promising tool for the treatment of neurodegenerative disorders that has proven to be safe and, in some cases, efficacious in early clinical trials. The delivery of genes of interest into the central nervous system (CNS) can be achieved through viral vector based approaches. Typically, this involves the coupling of transgene expression to a constitutively active promoter, driving high levels of transgene expression. However, this poses a limitation for use in the patient setting, where high expression levels of transgenes can result in unwanted adverse effects. Therefore, approaches in regulating gene expression are imperative. A number of the challenges faced with regulating gene expression can be overcome by the utilisation of a novel molecular switch developed previously in our laboratory. This system is dependent on the activation of proteases in response to endogenous pathological stimuli, enabling transgene expression to be achieved selectively in cells that are unhealthy. This thesis has focused on the optimisation and refinement of certain components comprising the novel molecular switch. Firstly, optimisation of the ARF5 transcription factor switch for future adeno associated viral vector (AAV)-mediated in vivo applications was conducted by assessing the transcriptional activity of truncated forms of the ARF5 protein in driving an enhanced green fluorescent protein (eGFP) reporter gene expression. The conduction of immunocytochemical, western blot and fluorescence-activated cell sorting (FACS) analysis revealed that the current ARF5 transcription factor switch could be truncated by 70%, whilst maintaining the same degree of transgene expression. In the final study, the evaluation of the efficiency of miRNA therapeutic cassettes in silencing huntingtin (htt) protein expression in an in vitro Huntington’s disease (HD) model was conducted. While, we were unable to conclude with any certainty the efficacy of miRNA-mediated silencing of htt, this set of experiments provided insights for directing future experiments. Collectively, these results provided the groundwork for the further optimisation and refinement of the novel molecular switch. Ultimately, broadening its applicability in the future to develop a regulated gene therapy approach for HD and other neurodegenerative diseases, involving calpain/caspase mediated pathogenesis.