Hazel Painter, Merton College, 2007.
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), leading to decreased chloride transport and increased sodium absorption across the airway epithelium. This causes dehydration of the airway surface liquid and reduced mucociliary clearance, resulting in chronic pulmonary infection and inflammation. Current gene therapy strategies, involving the introduction of a functional copy of CFTR into the airway epithelial cells, have so far been inefficient. Messenger RNA (mRNA) was investigated as a novel non-viral vector for CF lung gene therapy. Due to its cytoplasmic site of expression, mRNA avoids the nuclear membrane, postulated as a major barrier to gene transfer in the non-dividing cells of the airway epithelium. Reporter gene expression following administration of naked (uncomplexed) EGFP-mRNA was observed in the respiratory epithelial cells of the mouse nose. Transgene expression occurred at a lower level and in more cells than after naked plasmid DNA (pDNA) delivery. This may provide a level and distribution of transgene expression similar to the normal physiological expression of CFTR in the lung. The ability to repeatedly administer a therapeutic transgene without loss of efficacy is essential for long-term CF gene therapy, due to the natural turnover of transfected airway epithelial cells. Reporter gene expression in the mouse nose from mRNA encoding luciferase was transient, but readministration was possible at least four times (7 day intervals between dosing) without a reduction in luciferase activity. Using a vibrating-mesh nebuliser, aerosolisation of naked mRNA was possible with limited degradation, a requirement for topical delivery to the lung. However, transgene expression was low after naked mRNA delivery to the mouse lung via intranasal instillation. This appeared to reflect the presence of RNases in the lung surface liquid, as demonstrated by degradation of mRNA after incubation with bronchoalveolar lavage fluid (BALF). However, mRNA could be protected from degradation in BALF by incorporating modified nucleotides into the transcript, which may provide a means for successful delivery of naked mRNA to the lung. An alternative strategy using RNAi against the epithelial sodium channel, ENaC, was also investigated in order to target sodium hyper-absorption directly. Like mRNA, delivery of siRNA avoids the need for nuclear entry and thus may allow efficient knockdown of its target mRNA in non-dividing cells. No reduction in ENaC mRNA was observed following nasal delivery of naked siRNA against any ENaC subunit. An alternative delivery strategy was employed in the form of hydrodynamic tail vein injection, which, using pDNA, resulted in greater transgene expression in the lung than administration via direct instillation. Knockdown of ENaC α and β subunits was observed in the lung following delivery of low concentrations of naked siRNA using this technique. Together, these studies demonstrate proof-of-principal that naked mRNA or siRNA can be successfully delivered to the airways in the mouse model and, with further development, RNA-based vectors may provide an alternative to current non-viral CFTR gene transfer strategies.