Lawton, A. E., Sumner-Jones, S. G., Varathalingam, A., Davies, L. A., Hyde, S. C. & Gill, D. R.
Molecular Therapy, 9, S55Download Back
Non-viral vectors are being developed for gene replacement therapy for the lung disease associated with Cystic Fibrosis (CF). The majority of CF gene transfer studies have utilised viral promoters such as CMV that result in high level transgene expression, but may become attenuated in vivo leading to rapid loss of expression. Previously we have used the endogenous polyubiquitin promoter UbC to overcome promoter attenuation in the mouse lung (Gill et al, Gene Ther. 8:1539). In addition to maintaining persistent transgene expression in vivo, the choice of promoter can affect the cell specificity of gene expression. In CF the target cells are airway epithelial cells. The use of a cell-specific promoter could maximise gene transfer efficacy and reduce potentially deleterious effects of expression in non-relevant cell types. Previously, transgenic mice expressing EGFP from the FoxJ1 promoter have been shown to express in ciliated cells lining the respiratory epithelium of the nose and lung (Ostrowski et al, 2003, Mol. Ther. 8:637). In this study, a 1kb DNA fragment containing the FoxJ1 promoter sequence was amplified and used to replace the CMV promoter fragment in the luciferase expression plasmid pCIKLux to generate plasmid pFIKLux. Luciferase expression was confirmed in vitro following transient transfection into HEK293T cells (pCIKLux: 169.8 ± 54.1 RLU/µg; pFIKLux: 208.7 ± 32.5 RLU/µg). To evaluate expression in vivo, female BALB/c mice (6-8 weeks) were dosed with 100µg of either pCIKLux or pFIKLux in 150µl water (n=6) via intranasal instillation. In the lungs, mean luciferase activity (24 hours post-dose) was similar from pCIKLux (27.5 ± 19.1 RLU/mg) and pFIKLux (8.6 ± 4.8 RLU/mg, ANOVA, p=0.3427). Luciferase expression was also similar in the nasal epithelium following nasal perfusion (15min) of 200µg of pCIKLux or pFIKLux in 100µl water (pCIKLux: 24.8 ± 9.5 RLU/mg; pFIKLux: 30.5 ± 18.9 RLU/mg). A plasmid expressing Enhanced Green Fluorescent Protein (EGFP) under the control of the FoxJ1 promoter was constructed to investigate the cell specificity of transgene expression, following delivery to the mouse nose and lung. Whole mouse heads were fixed in paraformaldehyde, decalcified and prepared for cryosection. As a control, delivery of adenovirus expressing EGFP from the CMV promoter (2 x 1011 viral particles per mouse) resulted in cells of the olfactory epithelium (sustentacular cells, olfactory receptor cells) being positive 24hours post-dose. No EGFP positive cells lining the nasal cavity, were observed following delivery of plasmid vectors expressing EGFP. One day after lung delivery of 300µg of pFIKEGFP in 150µl water (n=2) the lungs were inflated, fixed with paraformaldehyde and individual lung lobes prepared for cryosection. 180 sections from each animal were examined using fluorescence microscopy, which revealed small numbers of GFP positive cells lining the airway epithelium. No GFP positive cells were observed in the lung parenchyma. Previously, lung administration of pEGFP-N1 (CMV promoter) revealed GFP positive cells in the both conducting airway epithelial cells (approx. 79%) and parenchymal cells (21%). Antibody co-localisation studies are underway to confirm the epithelial cell types. These studies indicate that reporter expression from the FoxJ1 promoter may not be greatly reduced compared to the 'strong' viral CMV promoter; and that cell-specific expression may help restrict transgene expression to the airway epithelium.