Repeated Exposure to pDNA/PEI Aerosols Results in Minimal Detectable Toxicity in the Mouse Lungs . (2009)

Nunez-Alonso, G. A., Davies, L. A., Bazzani, R. P., Cornish, N., Zhu, J., Alton, E. W. F. W., Hyde, S. C. & Gill, D. R.

Molecular Therapy, 17, S123

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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, successful gene therapy is likely to require repeated application of gene transfer agents (GTAs) to the small airways of the lung and nebulisation may be the most acceptable delivery system. The cationic polymer 25kDa branched polyethyleneimine (PEI) has been shown to retain transfection efficiency following aerosolisation. However, the direct instillation of pDNA/PEI complexes into rodent lungs is associated with significant inflammatory responses and concerns remain about the effects of repeated exposure of pDNA/PEI aerosols. To investigate this, female BALB/c mice received multiple aerosols of PEI complexed with the luciferase expression plasmid pCIKLux. Groups of n=6 mice received between one and 10 aerosols containing 20 ml of pCIKLux at 0.2mg/ml (N:P of 10:1) at weekly intervals and toxicity was assessed in all animals 24 hr after the final exposure, compared with untreated animals (naïve) and animals receiving 100µl of pCIKLux/PEI by intra-nasal instillation (PEI instilled). Robust gene expression was detected in the lung after a single aerosol exposure (41.3±10.0 RLU/mg) at levels equivalent to the PEI instilled group (21.8±5.9 RLU/mg) (p=0.12). Aerosol treated mice demonstrated equivalent weight gain compared with naïve mice over the course of the study. However, PEI instilled mice were noticeably less active in the 24 hr period after treatment and demonstrated a significant weight loss (7.8±0.4% of body weight) over the same period. Histological analysis of lung sections revealed no inflammatory changes in any of the PEI aerosol treated mice. However significant changes were observed in PEI instilled mice, with localised destruction of alveolar architecture associated with infiltrating inflammatory cells including macrophages, lymphocytes and neutrophils. In contrast similar numbers of inflammatory cells were observed in the BALF from the PEI aerosol, PEI instilled and naïve groups of mice. BALF was also analysed using a high-throughput multiplex assay for a panel of 23 cytokines (Bio-Plex, Bio-Rad). Again, no elevation of BALF cytokines was observed in any animal exposed to PEI aerosols although significant increases in the pro-inflammatory cytokines MCP-1, G-CSF, IL-6 and IL12p40 were detected in PEI instilled mice. These data demonstrate that whilst instillation of pDNA/PEI complexes into the mouse lung is associated with a robust inflammatory response, aerosol delivery of identical formulations results in equivalent gene expression in the absence of any detectable inflammatory changes, even after repeated exposure. This would suggest that delivery methodology plays an important role in the toxicity of PEI in the lung.

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