Davies, L. A., Hyde, S. C. & Gill, D. R.
Molecular Therapy, 13, S267Download Back
Plasmid DNA (pDNA) complexed to the cationic polymer polyethyleneimine (PEI) has been shown to retain transfection efficiency following aerosolisation and as a result, PEI has become a popular choice of gene transfer agent for lung gene therapy. Development of PEI for clinical applications has been hampered by the relatively low pDNA/PEI concentrations that can be prepared in the laboratory - typically less than 0.4mg/ml. We have investigated the use of a large-scale ultrafiltration protocol to generate stable concentrated pDNA/PEI formulations in excess of 8mg/ml and we have tested these formulations in a mouse lung model. Plasmid pCIKLux expressing the firefly luciferase gene under the control of the cytomegalovirus immediate/early promoter was complexed with branched 25kDa PEI (N:P of 10:1) at an initial concentration of 0.2mg/ml. This formulation was concentrated using a pressurised ultrafiltration cell incorporating a 100kDa cut-off cellulose membrane to produce final formulations containing 1,2,4 and 8mg/ml pDNA/PEI complexes. Following concentration, pCIKLux/PEI formulations were administered to the lungs of female BALB/c mice (8-16wks) via nasal instillation (100µl) or whole body aerosol exposure (10ml aerosol) and luciferase expression was determined in whole lung homogenates 24hrs later. Although instillation of pCIKLux/PEI formulations at 1mg/ml resulted in good levels of luciferase gene expression (71.1±16.7 RLU/mg protein), the observed expression was not significantly different from that achieved following instillation of standard 0.2mg/ml formulation (41.6 ±10.4 RLU/mg)(p=0.35 ANOVA). In addition, instillation of complexes to the mouse lung was associated with considerable dose-dependent toxicity, manifested by weight loss, histological changes and elevated neutrophil levels in bronchoalveolar lavage fluid (BALF). Instillation of pDNA/PEI complexes at 1mg/ml was associated with high mortality and higher concentrations were not assessed. In contrast, aerosol delivery of concentrated pDNA/PEI formulations to the mouse lung resulted in high levels of reporter gene expression with minimal associated lung toxicity. Aerosol delivery of pCIKLux/PEI at 1mg/ml produced luciferase expression at 133.2 ± 11.2 RLU/mg compared to 18.8 ± 1.9 RLU/mg for standard 0.2mg/ml formulations (p<0.0005 ANOVA). Higher concentrations resulted in increased luciferase expression at 2mg/ml (215.5 ±18.9 RLU/mg), 4mg/ml (274.0 ±39.2 RLU/mg) and even 8mg/ml (310.3 ± 55.5 RLU/mg) although expression did not increase in a linear fashion relative to the delivered dose. Minimal evidence of lung toxicity was observed following aerosol delivery with moderate weight loss and a slight elevation of BALF neutrophil numbers observed only at doses of 4mg/ml and above. These results demonstrate that viable concentrated pDNA/PEI formulations can be produced which generate high levels of reporter gene expression and minimal toxicity in the mouse lung following aerosol delivery.