Human Gene Therapy, 20 4 403
The British Society of Gene Therapy Annual Conference, London, 2009
Clinical studies are underway for the aerosol delivery of Genzyme Lipid GL67A complexed with plasmid DNA (pDNA) to the lungs of patients with cystic fibrosis (CF). The clinical plasmid utilises the ubiquitously expressing hCEFI (human Cytomegalovirus enhancer/elongation factor 1alpha) promoter to express the CFTR protein. This plasmid and a similar luciferase (Lux) expressing version generated persistent high levels of gene expression (> 8 weeks) following aerosol delivery of GL67A/pDNA to the mouse lung. However, we are also investigating promoters that have the potential to give tissue-specific CFTR gene expression, including a mini CFTR promoter encompassing the minimum sequence from the 5' region upstream of the CFTR gene able to provide promoter activity and confer cell-type specific expression in vitro (Chou et al. 1991, J. Biol. Chem, 266:24471). The promoter was cloned into our 4th generation CpG-free plasmid backbone in either a Native (promoter only) or Enhanced (plus human CMV enhancer) form. Versions of the Native and Enhanced plasmids with the human CFTR transgene or the luciferase reporter gene were constructed and both expressed significant levels of luciferase or CFTR mRNA in cell culture. CFTR protein activity was confirmed by iodide efflux. When gene expression from the Native and Enhanced plasmids was compared with expression from the synthetic hCEFI promoter following direct instillation into the mouse lung (80µg pDNA/100µl, BALB/c, n=6), Lux activity from the Enhanced plasmid gave 21% and 12% hCEFI levels at d1 and d14 respectively. These plasmids will also be assessed in human Air-Liquid Interface cultures to compare levels of expression in differentiated respiratory epithelia.