Specific genetic modifications in the CNS - Cell specific expression and gene specific regulation

Abstract: Gene therapy is a promising therapeutic tool for many diseases of the CNS. Lentiviral vectors are particularly attractive since this vector type can transduce both dividing and non-dividing cells, have a relatively large cloning capacity, can sustain long-term transgene expression and have low immunogenicity. The specificity of the vector is however important to avoid potential off-target effects and make the treatment as safe and beneficial as possible. The promoter plays a large part in the specificity of the vector. In this thesis we describe two approaches to find disease-specific promoters that can be used in gene therapy vectors for treatment of Parkinson’s disease. Another way to restrict transgene expression is by post-transcriptional regulation. This can be achieved by introducing miRNA target sites in the transgene cassette. We describe such an approach to restrict transgene expression to resident microglia in the brain. Perturbation in the GABAergic signalling is present in Parkinson’s disease and several other disorders of the CNS. Glutamic acid decarboxylase is the rate-limiting enzyme in the synthesis of GABA and the GABA output of a neuron may be altered when the level of GAD present in the cell is modified. In this thesis we show two different approaches to specifically regulate the expression of the endogenous GAD67 gene. GAD67 is one of the two isoforms of glutamic acid decarboxylase present in the brain. The first approach uses a customised zinc-finger based transcription factor to upregulate GAD67 while the second approach use a synthetic microRNA to downregulate GAD67. Both of these tools could potentially be used in disease modifying treatments of diseases where aberrant GABAergic signalling is present.