Studies on the Persistence of Transgene Expression in the Murine Airways

Ian Pringle, Trinity College, 2002.

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Non-viral gene therapy is being considered as a treatment for cystic fibrosis (CF). In clinical studies and in studies using the mouse airways as a model, current formulations result in only transient transgene expression. A number of reasons for this have been proposed including the loss of vector DNA from cells and transcriptional silencing of plasmid DNA (pDNA). The aim of these studies was to investigate why transgene expression from non-viral vectors is transient. Delivery of pDNA vectors encoding the luciferase (Lux) reporter gene to the mouse airways was used as a model system and its persistence in the mouse lungs was investigated by using quantitative PCR and Southern hybridisation. Results showed that intact pDNA persisted in the mouse lung in the absence of any detectable Lux activity. The de novo methylation of pDNA in the lung was investigated as a potential cause of this initial finding but the results suggested that pDNA does not become de novo methylated in the mouse lung. Therefore processes other than the loss of pDNA from the lung or the de novo methylation of pDNA vectors must be responsible for the transient reporter gene activity. The CpG response, an inflammatory response caused when mammalian cells are exposed to pDNA has been implicated in the silencing of pDNA vectors. The partial methylation of pDNA was investigated as a mechanism of overcoming this effect. Partially methylated vectors were produced which had Lux activity comparable to unmethylated pDNA in cell culture studies. The CpG response from these vectors was then investigated by measuring the inflammatory cytokine production from cultured mouse splenocytes following incubation with methylated and unmethylated pDNA. However, no reductions in cytokine production were observed from the partially methylated pDNA relative to the unmethylated pDNA. This suggested that partial methylation of pDNA vectors was not an effective technique for overcoming the CpG response from non-viral based gene therapy vectors treatment of CF.

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