Molecular Therapy, 16 S160
The American Society of Gene Therapy Annual Conference, Boston, 2008
The UK Cystic fibrosis gene therapy consortium will commence gene therapy trials involving delivery of cationic lipid based gene transfer agents to the lungs of over 100 CF patients in the UK.
Topical aerosol delivery of a CpG-free plasmid expressing the CF gene product (hCFTR) complexed to the cationic lipid GL67A will be achieved using a clinical jet nebuliser device. Careful selection of a suitable nebuliser is required to ensure minimal degradation of shear-sensitive pDNA molecules and generation of aerosols with appropriate size characteristics for targeted aerosol delivery to the lung (mass median aerodynamic diameter (MMAD) 2-4 m).
We have investigated the suitability of 5 commercially available jet nebulisers, the Pari LC+, Sprint, Sprint Junior, Sprint Star and the Trudell AeroEclipse II breath-actuated nebuliser for aerosol delivery of pDNA/GL67A complexes. In contrast to un-complexed pDNA, aerosolisation of pDNA/GL67A formulations (8 mM:6 mM in sterile water) with each nebuliser was associated with minimal plasmid degradation and retention of biological activity was demonstrated following aerosol delivery of 25 mg of a luciferase expression plasmid to the lungs of BALB/c mice (n=6 per group) using a whole body aerosol exposure apparatus. Robust levels of gene expression were detected in all treated mice with expression ranging from 13.8 3.6 RLU/mg with the LC+ up to 73.1 7.8 RLU/mg using the Sprint Junior. Aerosol size characteristics for each nebuliser were determined following inertial impaction of aerosol in a chilled (7-8C) Next Generation pharmaceutical Impactor (NGI) operating at 15 L/min.
As expected, the MMAD for each nebuliser was highly dependent upon the operating pressure used to generate aerosol with the smallest droplets produced at 50 psi - the highest operating pressure that could be assessed safely. At 50 psi, aerosols with suitable MMAD properties for human delivery were produced by the Sprint Junior (3.43 0.07 m), Sprint Star (3.37 0.09 m) and the AeroEclipse II (3.40 0.1 m) nebulisers. However the Sprint Star nebuliser was prone to sputtering with pDNA/GL67A formulations and was excluded from further studies. Assessment of aerosol delivery rate was performed using a human breath simulator operating with a sinusoidal breathing pattern at 15 breaths/min, an inspiratory: expiratory ratio of 1:1 and a tidal volume of 500 ml.
Aerosol delivery of pDNA/GL67A formulations using the Sprint Junior nebuliser was faster (235 20 l/min) than the AeroEclipse II nebuliser (170 6 l/min) but the efficiency of the breath-actuated AeroEclipse II was significantly higher (83 2% compared to 55 1%) as determined by collection of aerosol on the inspiratory arm of the breathing circuit.
These data suggest that both the Sprint Junior and AeroEclipse II nebulisers would be suitable for aerosol delivery of pDNA/GL67A formulations for CF gene therapy studies in humans.