Local and Systemic Inhibition of Lung Tumor Growth after Liposome Mediated mda-7/IL-24 Gene Delivery

Compacted DNA nanoparticles consist of single molecules of plasmid DNA condensed with polyethylene glycol (PEG)-substituted lysine polymers. Using a 30-mer lysine polymer conjugated to 10 kDa PEG, we recently conducted a dose escalation intranasal Phase I clinical trial in cystic fibrosis (CF) subjects. No adverse events were attributable to the clinical trial material, and 8 of 12 subjects had evidence of partial to near complete CFTR chloride channel reconstitution as monitored by nasal potential difference measurements of an isoproterenol response in chloride-free buffer containing amiloride. To prepare compacted DNA nanoparticles in a formulation suitable for whole lung dosing, we have conducted aerosol optimization studies using the Aerogen Aeroneb Pro® and Pari LC Star™ devices. Using a DNA concentration of 2 mg/ml, aerosols of compacted DNA were readily generated using both devices. The particle size distribution was determined using cascade impactors and aerosol mists were collected in saline-filled impinger tubes for structural and biological studies. Using a flow rate of 6 L/min for both devices, the Aeroneb Pro® generated an aerosol having a mass median aerodynamic diameter (MMAD) of 4.0 μm with a geometric standard deviation (GSD) of 2.1 μm, whereas the Pari LC Star™ generated an MMAD of 2.2 μm with a GSD of 2.3 μm. The solution use rate using the Aeroneb Pro® device was 0.350 ml/min with a DNA aerosol concentration of 0.055 mg/L, whereas the Pari LC Star™ use rate was 0.247 ml/min with a DNA aerosol concentration of 0.030 mg/L. The lower DNA aerosol concentration generated by the Pari LC Star™ device reflects, in part, the lower solution use rate. However, the aerosol DNA concentration remained constant over time using the Pari LC Star™ device, whereas it increased from approximately 0.03 to 0.07 mg/L using the Aeroneb Pro® generator. To evaluate structural integrity, saline impinger samples were concentrated to ~200 to 300 μg/ml using centrifugal concentrators (100,000 MW cut-off) and assayed by electron microscopy (DNA nanoparticle size and shape), turbidity and sedimentation assays (colloidal stability), serum stability tests (protection of DNA from nucleases), and gel analysis (DNA integrity). The post-aerosol samples for both devices retained structural integrity, comparable to the results observed for the pre-aerosol material. To monitor biological activity, pre- and post-aerosol compacted DNAs encoding luciferase were used to transfect HepG2 human hepatoma cells in culture and the lungs of Balb/C mice. In both cases, luciferase activities per mg protein for the pre- and post-aerosol samples were identical. In summary, aerosols of compacted DNA have been generated that have size distributions appropriate for proximal lung delivery and which retain structural integrity and biological activity. In progress are IND-directed preclinical aerosol lung efficacy, toxicology, and DNA biodistribution studies in support of a planned lung aerosol clinical trial in CF subjects.