Pediatric Pulmonology, 40 S28 291
The North American Cystic Fibrosis Conference, Baltimore, 2005
As part of the UK Cystic Fibrosis Gene Therapy Consortium, we are aiming to identify non- viral gene transfer agents (GTAs) that are candidates for delivery of CFTR to human airways. In addition to GTAs developed within the Consortium, we have evaluated commercially available and proprietary GTAs from academic and industrial collaborators. GTAs evaluated include examples of cationic lipids/liposomes, cationic polymers and DNA condensing or cell targeting peptides. The GTAs are screened first in the mouse nose using a luciferase reporter expression plasmid, before selecting promising formulations for subsequent CFTR function testing in the CF transgenic mouse nose and for clinically relevant aerosol delivery to the sheep lung. The initial testing in the wild type mouse nose involves the perfusion of 100ml of the GTA into the nostrils of C57BL6 mice over 15 minutes, or 75 minutes to maximise GTA contact time with the epithelium. Our studies are powered at 0.8- 0.95 with n=6-8 per group. After 24 hours, the nasal epithelium is removed and divided into proximal (containing mainly respiratory cells) and distal epithelium (containing mainly olfactory cells). Luciferase and protein assays (with intra- and inter-assay variability <= 10%) are performed to determine reporter activity that was between 5- and 20-fold greater in the respiratory epithelium for all GTAs tested in this way. Once a suitable component ratio had been established, the GTAs were tested under standardised, comparable conditions, including a dose response between the Maximum Feasible Dose (MFD) and one tenth of the MFD. All GTAs are evaluated with the same plasmid (pCIKLux) resulting in CMV- mediated peak expression at 1 or 2 days post-administration. As previously observed in the lung, CMV-mediated expression is transient in the nose, falling to background within 1 week, apparently irrespective of the non-viral GTA tested. However, when utilising a novel plasmid developed for clinical studies, in which the human promoter polyubiquitin C (UbC) directs gene expression in the context of a CpG reduced backbone, persistent luciferase activity can be maintained for up to 28 days in some cases. Luciferase expression for one particular GTA increased from 60-fold over naive levels on day 1 (p=0.0004) to 600-fold on day 14 (p< 0.0001). Currently more than 25 non-viral GTAs have been tested in this core facility; the results from three of the most fully characterised products will be presented to demonstrate the iterative process required to optimise airway specific gene expression.