143. Use of Ciliated Cell Specific Promoter FoxJ1 in Gene Transfer Vectors for the Airway Epithelium
Authors: Anna E. Lawton, Stephanie G. Sumner-Jones, Anusha Varathalingam, Lee A. Davies, Stephen C. Hyde, Deborah R. Gill
Journal: Molecular Therapy (2004)

Non-viral gene therapy vectors are being developed for Cystic Fibrosis (CF) lung disease. Most CF gene transfer studies employ viral promoters, eg. CMV, that result in high level transgene expression, but become attenuated in vivo leading to rapid loss of expression. Previously we used endogenous polyubiquitin promoter UbC to overcome promoter attenuation in mouse lung (Gill et al, Gene Ther. 8:1539). As well as maintaining persistent transgene expression in vivo, the choice of promoter affects cell specificity of gene expression. In CF the target cells are airway epithelial cells. The use of a cell-specific promoter could boost gene transfer efficacy and reduce potentially deleterious effects of expression in irrelevant cells. Transgenic mice expressing EGFP from the FoxJ1 promoter have been shown to express in ciliated cells in the respiratory epithelium of nose and lung (Ostrowski et al, 2003, Mol. Ther. 8:637). In this study, a 1kb DNA fragment containing the FoxJ1 sequence was used to replace the CMV promoter in luciferase (lux) plasmid pCIKLux, generating pFIKLux. Lux expression was confirmed in vitro following transient transfection into HEK293T cells (pCIKLux: 169.8 54.1 RLU/g; pFIKLux: 208.7 32.5 RLU/g). To evaluate expression in vivo, female BALB/c mice (6–8 weeks) were dosed with 100g of pCIKLux or pFIKLux in 150l water (n=6) via intranasal instillation. In lungs, mean lux 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). Lux expression was also similar in nasal epithelium following nasal perfusion (15min) of 200g of pCIKLux or pFIKLux in 100l 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 1011 viral particles per mouse) resulted in cells of the olfactory epithelium (sustentacular, olfactory receptor) being positive 24hours post-dose. No EGFP+ve cells lining the nasal cavity were observed following delivery of plasmid vectors expressing EGFP. 24hr after lung delivery of 300g of pFIKEGFP in 150l water (n=2) lungs were inflated, fixed and cryosections taken. 180 sections from each animal were examined using fluorescence microscopy, revealing low numbers of GFP+ve cells lining the airway epithelium. No GFP+ve cells were observed in lung parenchyma. Previously, lung administration of pEGFP-N1 (CMV promoter) revealed GFP+ve 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 CMV promoter, and that cell-specific expression may help restrict transgene expression to the airway epithelium.