An investigation into persistent transgene expression from plasmid vectors in the lung

Gene therapy for chronic lung diseases will require vectors capable of persistent transgene expression in the absence of inflammation but few promoters have been identified that can fulfil both of these requirements. The development of plasmids devoid of CG dinucleotides (CpGs) has resulted in markedly reduced levels of pro-inflammatory cytokines. Furthermore, a novel synthetic enhancer/promoter, termed hCEFI, has been shown to direct high levels of sustained transgene expression in the murine lung, following aerosol delivery of non-viral formulations. A CpG-free plasmid containing the hCEFI enhancer/promoter has been evaluated in clinical trials for cystic fibrosis lung gene therapy. The aim of this thesis was to investigate the reasons for successful persistent expression achieved with this plasmid. An understanding of these reasons is also useful for the development of new gene therapy treatments. Cell culture models did not recapitulate the transgene expression profiles obtained in vivo; therefore, a mouse aerosol model of lung gene transfer was utilised. New variants of the hC enhancer present in the hCEFI promoter were constructed and tested in vivo. Studies demonstrated that a minimal enhancer sequence, comprising the final 93 bp of the full-length 302 bp hC enhancer, was necessary and sufficient for mediating persistent and high levels of transgene activity in the mouse lung. Support for the inclusion of multiple copies of the minimal enhancer to increase transgene activity was also obtained. Observations in a mouse model for gene transfer to the liver showed that the minimal enhancer could not mediate transgene activity at the same level as the full-length hC enhancer, indicating that the minimal enhancer does not uniformly direct transgene activity but may be specific to the target organ. An analysis of predicted transcription factor binding sites was performed to support such tissue-specific differences. Overall, these studies demonstrate that only a very small portion of an enhancer is required to direct high-level activity in the lung. This has implications for the future design of both non-viral and viral vectors for use in gene therapy applications.