Aerosol Characteristics of DNA/lipid Formulations for Gene Therapy Clinical Studies.

Gill DR, Nunez-Alonso GA, McLachlan G, Cheng SH, Scheule RK, Hyde SC, Davies LA

Pediatric Pulmonology, Vol 31, 307

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The North American Cystic Fibrosis Conference, Orlando, 2008

Viral and non-viral gene transfer agents (GTAs) are being evaluated for treatment of CF lung disease, but assessment in animal models that deliver a bolus of fluid to the lung via tracheal instillation or nasal sniffing can report misleading toxicity and efficiency. Instead, evaluation of GTAs requires aerosol delivery in appropriate animal models. The UK Cystic Fibrosis Gene Therapy Consortium has compared several non-viral GTAs following aerosol delivery to mouse and sheep lung and has selected cationic lipid GL67A for further clinical studies.

To select a nebuliser for optimal delivery in the clinic, five commercially available jet nebulisers Pari LC+, Sprint, Sprint Junior, Sprint Star and the Trudell AeroEclipse II breath-actuated nebuliser were compared for aerosol delivery of pDNA/GL67A formulations (8 mM:6 mM in sterile water). In contrast to un-complexed (naked) pDNA, aerosolisation of DNA/lipid was associated with only minimal plasmid degradation with all five nebulisers. Furthermore, robust Luciferase reporter expression was demonstrated in the lung following aerosol delivery to BALB/c mice (25mg pDNA; n=6 per group) using a whole body aerosol chamber, with results ranging from 13.8 ± 3.6 RLU/mg with the Pari LC+ up to 73.1 ± 7.8 RLU/mg using the Sprint Junior.

In addition to maintaining the integrity of the formulation, nebulisers must generate aerosol droplets with mass median aerodynamic diameter (MMAD) suitable for delivery to the small airways of the CF lung (estimated 2-4 µm). This was confirmed for each nebuliser following inertial impaction of aerosol in a chilled (7-8?C) Next Generation pharmaceutical Impactor (NGI) operating at 15 L/min. As expected, the MMAD for each nebuliser was dependent upon the operating pressure with the smallest droplets produced at 50 psi; the Sprint Junior (3.43 ± 0.07 µm), Sprint Star (3.37 ± 0.09 µm) and the AeroEclipse II (3.40 ± 0.1 µm) nebulisers generated aerosols with MMAD suitable for human delivery The Sprint Star nebuliser was prone to "sputtering" with pDNA/GL67A formulations and was excluded from further studies.

Using a human breath simulator (sinusoidal breathing pattern at 15 breaths/mi; an inspiratory: expiratory ratio of 1:1; tidal volume of 500 ml) the aerosol delivery rate of pDNA/GL67A using the Sprint Junior nebuliser was faster (235 ± 20 µl/min) than the AeroEclipse II nebuliser (170 ± 6 µl/min). However the efficiency of the breath-actuated AeroEclipse II was significantly higher (83 ± 2% compared with 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 in CF gene therapy clinical studies.