Davies, L. A., Hyde, S. C. & Gill, D. R.
Molecular Therapy, 11, S137Download Back
Gene therapy for many chronic diseases will require long-term gene expression and/or repeated administration. We are investigating gene transfer to the terminally differentiated epithelial cells of the airways, the target for the treatment of cystic fibrosis lung disease. Unlike many viral vectors where repeat administration may be severely compromised by neutralizing antibodies, non-viral vectors such as cationic polymers and cationic lipids are not associated with significant immune responses in the lung and may provide a more appropriate alternative for long term gene therapy. We have utilised aerosol delivery of PEI/DNA complexes to the lungs of mice as a model to investigate gene expression following repeat administration of this non-viral gene transfer agent in vivo. Female BALB/c mice housed in a whole body aerosol exposure chamber were exposed repeatedly to aerosols containing 25kDa branched PEI (Sigma, Poole, UK) complexed to plasmid DNA expressing the firefly luciferase gene under control of the cytomegalovirus immediate/early promoter. A total of 4 aerosol doses were delivered at intervals of 7 days. Following each aerosol exposure, groups of mice were sacrificed after 1 day and 7 days and luciferase expression was assayed in whole lung homogenates. Following a single exposure to PEI aerosol, robust luciferase expression at 68.0 ± 10.3 RLU/mg (mean ± SEM) protein was detected in the lungs of treated mice at 24 hrs after exposure. The observed expression was transient and had fallen to 4.7 ± 1.2 RLU/mg by the time of the second aerosol exposure at 7 days. Following a second aerosol, high levels of luciferase expression were again detected in the lungs of treated mice but maximal expression reached only 49.3 ± 3.8 RLU/mg at 24 hrs (p=0.027 compared to initial dose using ANOVA/PLSD analysis). Further reductions in lung luciferase levels were observed following the third and fourth aerosol exposures where luciferase levels reached only 15.8 ± 1.9 RLU/mg (p<0.0001) and 13.3 ± 2.1 RLU/mg (p<0.0001) respectively. NaÔve mice exposed contemporaneously showed no equivalent fall in luciferase expression. Subsequent experiments to investigate the mechanism behind the observed fall in luciferase expression upon repeat administration of PEI/DNA showed that the effect was independent of the transgene used. In addition, prior exposure to aerosols containing PEI alone was sufficient to mediate the loss of transfection efficiency. These results demonstrate that repeat administration of 25kDa PEI/DNA aerosols to the lung is associated with a significant loss of transfection efficiency upon re-administration at intervals of 7 days. Experiments are currently underway to determine if the observed results represent a refractory period for effective aerosol re-administration and if longer dosing intervals may permit restoration of initial gene expression levels in this model system.