Mutliple Doses of Lipid Mediated Gene Therapy Nebulised to the Mouse Lung Show Robust and Sustained CFTR Expression.

Hyde SC, Sumner-Jones SG, Pringle IA, Cheng SH, Scheule RK, Alton EW, EW, Davies JC, Higgins T, Boyd AC, Innes AJ, Porteous DJ, Gill DR, Griesenbach U

Pediatric Pulmonology, Vol 46, Abstract 197

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The North American Cystic Fibrosis Conference, Anaheim, 2011

The nebulisation of multiple doses of a non-viral gene therapy formulation to the lungs of individuals with CF is being considered as a treatment for chronic CF lung disease. The success of such a strategy depends on the selection of a formulation capable of CFTR expression that is high-level, sustained, and can be repeatedly administered. Plasmid pGM169 is free of CG dinucleotides and contains the novel synthetic hCEFI promoter specifically designed for long-term expression of CFTR in the lung; when complexed with cationic liposome GL67A (supplied by Genzyme) CFTR expression lasts for several months in the mouse lung after a single nebulised dose (Hyde et al 2008 Nature Biotech 26:549). A clinical trial to assess the safety and CFTR expression of a single nebulised dose of pGM169/GL67A in 35 CF patients has just been completed (separate abstract). In order to progress into multiple-dose studies a GLP toxicology (separate abstract) and biodistribution study was performed in mice to evaluate the CFTR expression profile of up to 12 doses of pGM169/GL67A. Mice were exposed to aerosolised pGM169/GL67A at 2 weekly intervals for 0.5, 2 or 6 hours (Low, Medium and High dose groups respectively); mice exposed to air for 6 hours were used as controls. Groups of mice (males and females; n=4-10) were sacrificed after one, six and 12 doses and organs (lung, cervical lymphnodes, spleen, gut (proximal ileum), liver, kidney, testes/ovaries, and blood) analysed for the presence of plasmid DNA and/or vector-specific CFTR mRNA using quantitative RT-PCR. A significant dose-response was observed between duration of inhalation and the quantity of plasmid DNA present in the lungs 1 day after delivery of one, six and 12 doses (p<0.0001; Spearman correlation). Plasmid DNA remained detectable in the lungs of animals for up to 21 weeks after the final (High) dose. Levels of plasmid DNA in non-target organs were several orders of magnitude lower than the lungs at day 1. When CFTR mRNA was measured in the lungs, low levels were detected after a single dose in the Low and Medium groups, with increased signal in the High group (p<0.001; equivalent to ≥100% endogenous levels). Importantly, after 12 doses, a cumulative treatment effect was noted such that high mRNA levels were observed for all animals in all treatment groups. Robust levels of CFTR mRNA remained in the lung for at least 21 weeks after the final exposure. This is the first time that multiple nebulised doses of a lipid-based formulation have been shown to achieve a long-lasting and cumulative CFTR expression that can build with successive doses; importantly this confirms our clinical strategy to deliver multiple doses in order to maximise CFTR expression. In conclusion, these studies further support progression into our planned multi-dose clinical trial designed to generate clinical benefit in CF patients.