Pediatric Pulmonology, 33 S1 Abstract 275
The North American Cystic Fibrosis Conference, Baltimore, 2010
Aerosol delivery of gene transfer agents is the most practical route for CFTR based gene therapy for CF lung disease. When complexed with plasmid DNA (pDNA), the cationic polymer, 25kDa branched polyethyleneimine (PEI) retains transfection efficiency following aerosolisation and consequently is a popular non-viral choice for lung gene transfer.
Until recently, progress towards the clinical use of PEI was limited by the low pDNA concentration achievable in standard PEI formulations. However, we have shown that concentrated PEI (cPEI) formulations containing up to 8 mg/ml pDNA can be prepared by controlled ultrafiltration of pDNA/PEI complexes, leading to robust levels of gene expression in the lungs of mice and sheep following aerosol delivery. We have capitalised on this important development to further exploit the potential of cPEI aerosol delivery for use in the clinic, focusing on formulations that are physically stable and suitable for long-term storage following manufacture, an absolute requirement for clinical utility.
Plasmid DNA encoding firefly luciferase reporter gene was complexed with branched 25kDa PEI (N:P of 10:1) at an initial pDNA concentration of 0.2 mg/ml in sterile water and concentrated to 2.0±0.2 mg/ml using a pressurised ultrafiltration cell incorporating a 100kDa cut-off cellulose membrane. Complexes were filter sterilised and aliquots stored under nitrogen in glass vials at 4±2 °C for 0, 1, 2, 4, 8, or 21 weeks. Visual inspection revealed no evidence of aggregation or precipitation at any time-point. A small increase in particle size from 62.5±0.3 nm (mean±SEM) at 0 weeks to 65.1±0.3 nm at 21 weeks (p0.05 ANOVA & Tukey's multiple comparison test).
In conclusion, we have utilised physical characterisation and in vivo expression studies to investigate the stability of cPEI complexes, demonstrating that pDNA/cPEI formulations prepared by ultrafiltration are stable and can be stored for extended periods at +4ËšC with minimal loss of biological efficacy. This provides further evidence to support the potential of cPEI for clinical aerosol gene therapy to treat CF lung disease.