Davies, L. A., Seguela, A., Varathalingam, A., Hyde, S. C. & Gill, D. R.Download Back
The murine lung has been used as a model to evaluate of gene therapy vectors for cystic fibrosis lung disease. Following delivery of non-viral vectors to the adult mouse lung, we wished to identify the specific cell types expressing transgene. Reliable tissue processing techniques were developed that allowed direct visualisation of cells expressing the red-shifted variant of green fluorescent protein (EGFP). To examine gene expression from naked plasmid DNA, BALB/c mice (n=3) were dosed intranasally with 300µg of plasmid pEGFP-N1 and after 24 hours, 7µm cryosections were prepared from lung tissues. GFP positive cells were identified in all treated mice with the majority (79%) of these cells located in the epithelium of conducting airways and the remainder (21%) located in the lung parenchyma. Immunohistochemical analysis with a range of cell type specific antibodies revealed that the majority of these GFP positive cells were ciliated epithelial cells, thought to be the major target for cystic fibrosis gene therapy. For treatment of chronic lung disease it is likely that persistent transgene expression will be required. Previously, luciferase expression under the control of the human polyubiquitin (UbC) promoter was shown to persist for up to 6 months in the mouse lung; therefore we constructed similar plasmids to express EGFP from the UbC promoter (pUbEGFP). Following instillation of 300µg of naked DNA (pUbEGFP; n=3), similar levels of expression were observed to that seen with pEGFP-N1, containing the CMV promoter, with the majority (61%) of GFP positive cells found in the conducting airways. These data confirm that previously observed persistent transgene expression from the UbC promoter in the mouse lung is located in the epithelial cells lining the conducting airways, and suggests that the UbC promoter may be useful in plasmid vectors for planned clinical studies in cystic fibrosis patients.