Molecular Therapy, 13 S1 S267
The American Society of Gene Therapy Annual Conference, Baltimore, 2006
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.