Robinson, Michael James; (2000) Development of herpes simplex virus vectors with a neuronal-specific enolase promoter. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Gene therapy is a technique that may in the future allow the treatment of diseases either by allowing genetic defects to be corrected or to allow other gene products to be produced which will reduce the pathology of the disease. Herpes simplex virus type 1 has been identified as a potentially ideal vector for gene therapy in the nervous system e.g. for neurodegenerative diseases such as Parkinson's disease, due to a natural ability to infect neurones and its ability to establish lifelong latent infection in the host. Problems with HSV vectors have included its cytotoxicity, short-term transgene expression and a lack of cell-type specificity. This thesis will describe work carried out in order to produce an HSV vector, which allows long-term expression in a neuron-specific fashion. Disabled HSV vectors were produced which contained a 1.8kB region of the neuronal-specific enolase promoter linked to various viral latency associated transcript (LAT) regulatory sequences in order to drive expression of the β-galactosidase marker gene. These latency associated promoter (LAP) sequences have previously been shown to confer long-term expression characteristics on heterologous promoters. Initially the promoter constructs were inserted into the UL43 locus of the HSV vectors. When these vectors were tested in in in vitro and in vivo assays, only very low levels of non-specific expression from the NSE promoter were observed. A second series of recombinant vectors were made where both a full length and a minimal (255Bp) NSE promoter driving β-galactosidase were inserted downstream of the endogenous LAP promoters. When these vectors were tested in dorsal root ganglia in vivo, the minimal NSE promoter gave high levels of activity comparable to a control virus, containing the constitutive CMV promoter. This virus maintained expression of the exogenous gene for at least 90 days. Moreover, whilst high levels of expression occurred at the inoculation site (footpad) with the control virus, the minimal NSE-containing virus gave only very low levels of expression at this site, indicating cell-type specificity. In order to further test the cell-type specificity of the viruses, they were used to infect organotypic hippocampal slice cultures. Whilst the control virus infected all areas of the hippocampus in both neuronal and non-neuronal areas, the minimal NSE virus showed a pattern of expression restricted to the main neuronal layers of the hippocampus as determined by X-gal staining. Specificity of the minimal NSE promoter in these slice cultures was confirmed by immunohistochemistry. Therefore, the recombinant HSV vector which contains the 255Bp NSE promoter inserted downstream of the LAT region is capable of long-term expression in a neuronal specific manner.

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