Molecular Therapy, 15 S1 S407
The American Society of Gene Therapy Annual Conference, Seattle, 2007
Gene transfer to the airways is being investigated as a possible therapy for a variety of acute and chronic lung diseases. In the case of cystic fibrosis, any successful gene therapy is likely to require repeated application of gene transfer agents (GTAs) to the small airways of the lung and nebulisation is likely to be the most acceptable delivery system.
The cationic polymer polyethyleneimine (PEI) has been shown to retain transfection efficiency following aerosolisation and as such has become a popular choice for lung gene therapy. However, development of PEI as a viable aerosol gene transfer agent has been hampered by the relatively low vector 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 concentrated DNA/PEI (cPEI) formulations in excess of 8mg/ml.
Plasmid pCIKLux expressing the firefly luciferase gene 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 cutoff cellulose membrane to produce final formulations containing 1,2,4 and 8mg/ml DNA/PEI complexes. No aggregation of pDNA/PEI complexes was observed during ultrafiltration although concentration of complexes from 0.2mg/ml to 8mg/ml was associated with a small but significant increase in particle size from 81.9 ± 2.1 nm to 124.9 ± 4.9nm as determined by dynamic light scattering. A concomitant increase in particle surface charge from +28.8 ± 0.1 mV to +36.7 ± 0.1 mV was also observed. To investigate the viability of cPEI formulations in vivo these formulations were administered to the lungs of female BALB/c mice (8-16wks) via whole body aerosol exposure (10ml aerosol) and luciferase expression was determined in lung homogenates 24hrs later. Aerosol delivery of cPEI formulations resulted in dose-dependent luciferase expression with the highest levels (310 ± 55 RLU/mg) detected in mice exposed to aerosols containing 8mg/ml pCIKLux.
Concentrated PEI formulations were also assessed in the sheep lung - a more clinically relevant large animal model for lung aerosol delivery. Aerosol delivery of 20ml of standard 0.4mg/ml pCIKLux/PEI formulations (8mg total plasmid dose) to sheep (n=6) resulted in detectable lung luciferase expression at 0.61 ± 0.28 RLU/mg. Increasing the aerosol volume to 80ml (32mg plasmid) increased gene expression to 5.08 ± 2.18 RLU/mg but was associated with a significant increase in inflammatory neutrophils detected in the lung. In contrast, aerosol delivery of a similar 32mg plasmid dose with cPEI formulations (20ml pCIKLux/PEI at 1.6mg/ml) resulted in equivalent luciferase expression (8.00 ± 2.73 RLU/mg) with a significantly reduced neutrophil response.
These results demonstrate that viable concentrated DNA/PEI formulations can be produced by ultrafiltration and that these formulations can generate high levels of reporter gene expression with minimal toxicity in vivo following aerosol delivery.