Syahril Abdullah, Keble College, 2005.
Non-viral gene therapy is being considered as a potential treatment for chronic cystic fibrosis lung disease. In the murine airways, which have been used to model lung gene transfer, the resultant transgene expression is transient. One of the problems that hinders prolonged gene expression of pDNA gene transfer vectors in vivo is promoter attenuation. Pro-inflammatory cytokines, largely induced by the administration of the gene transfer vector, have been implicated in this phenomenon. This study investigated strategies designed to improve the persistence of transgene expression in the murine airways following delivery of cationic lipid GL67 complexed with pDNA. Three strategies were tested: (1) the incorporation of an insulator element to protect the promoter from attenuation, (2) the administration of an immunosuppressive drug to reduce pro-inflammatory cytokines, and (3) the modification of the pDNA construct itself. The addition of a putative insulator element, Ubiquitous Chromatin Opening Element (UCOE), 5' to a CMV promoter did not improve the duration of transgene expression. The administration of an immunosuppressive drug, dexamethasone, prior to pDNA delivery, only moderately reduced the pro-inflammatory cytokines with variable results on transgene activity. However, modification of the pDNA construct to fully deplete CpG sequences was shown to reduce the induction of pro-inflammatory cytokines and subsequently extend the duration of transgene expression up to 60 days post-administration although the precise mechanism is still questionable. These initial results have demonstrated that a potentially therapeutic level of transgene expression over an extended period may be achieved by the use of CpG-depleted plasmids in non-viral vectors.