Characterisation of a protease-dependent gene regulation system for gene therapy applications

Abstract

Gene therapy is a promising approach for the treatment of a wide range of chronic diseases. These therapies typically utilise strong, constitutively active promoters to drive high levels of recombinant protein expression. Unregulated gene expression is a key issue limiting clinical translation of promising gene therapies due to the potential for side-effects from excessive levels of protein that indiscriminately affect both diseased and non-diseased cells. Ideally gene expression would be regulated such that it would be quiescent under basal conditions and induced to a therapeutic level in at-risk cells only. This thesis describes the characterisation of a novel bicistronic autoregulatory gene expression system that relies on cell stress-induced activation of specific proteases to drive transgene expression. The functionality of this system was assessed in vitro in a model of cellular stress and in vivo in a neurotoxin model of Parkinson’s disease, a neurodegenerative disorder associated with aberrant activation of the specific proteases caspase-3 and calpain. The results showed that transgene expression under control of the gene regulation system was low under basal conditions and inducible in a concentration-dependent and temporal manner in response to cellular stress induced by the cytotoxin okadaic acid in vitro. Following adeno-associated viral vector (AAV)-mediated delivery of a green fluorescent protein (GFP) reporter gene under control of the regulatory system to the rat substantia nigra pars compacta, expression of GFP was induced in response to cellular stress initiated in the striatum. In the final study the protective efficacy of the anti-apoptotic gene X-linked Inhibitor of Apoptosis Protein (XIAP) under the control of this system was examined in comparison to constitutive XIAP expression in the 6-OHDA model. The results showed that regulating XIAP expression with this system afforded greater protection of dopamine neurons in comparison to control animals that express GFP under control of the regulatory system. In contrast, the extent of dopamine cell loss in animals injected AAV vectors expression XIAP under control of a constitutively active promoter was not different to the corresponding control GFP vector-injected rats. Together these results provided proof-of-principle evidence of the utility of this system for the treatment of brain disorders associated with increased activation of proteases.