(298b) Airway Gene Transfer and Intracellular Trafficking of Highly Compacted DNA Nanoparticles

Despite over two decades of cystic fibrosis (CF) gene therapy research, development and clinical testing, no patient has yet been cured. Poor gene transfer to the airway epithelium has been attributed to a number of cellular barriers, including poor cellular uptake across the apical membrane, unproductive intracellular trafficking, and inefficient nuclear import. Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of poly-L-lysine and 10 kDa polyethylene glycol (CK₃₀PEG_10k), have shown considerable promise in human gene therapy clinical trials in the nares, but efficacy has not been established in the CF lung. Starting with the clinically tested DNA nanoparticle system, we have systematically varied the PEG MW (from 10kDa to 2kDa) and poly-L-lysine architecture and studied their effects on particle morphology, cellular uptake and intracellular trafficking, and gene transfer to lung airways in BALB/c mice. In addition, we will discuss a correlation between our intracellular trafficking data and in vivo gene transfer results. Together, these results represent an important step toward the rational development of an efficient gene delivery platform for the lungs based on highly compacted DNA nanoparticles.