Development of non-viral systems for pulmonary nucleic acid delivery and gene editing

The fast-pace development of genome-editing technologies brings hope to provide a cure for genetic diseases fundamentally. However, the inefficient delivery of genome-editing systems including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-associated nuclease Cas9 (CRISPR/Cas9) still limits their clinical applications. Compared to systemic delivery, pulmonary delivery enables noninvasive and direct deposition of drug in lung, which has made pulmonary delivery promising for the treatment of many lung diseases including cystic fibrosis (CF). Gene-editing systems are also attractive via this route for enhanced regional targeting and avoidance of clearance and biological barriers that limit delivery efficiency of systemically administered therapeutics. The objective of the dissertation was to explore non-viral delivery platforms for pulmonary delivery of nucleic acids including DNA and messenger RNA (mRNA). In Chapter 1, a review of the development of delivery of genome-editing systems is provided. In Chapter 2, the development of a PEGylated chitosan carrier system for the delivery of CRISPR/Cas9 genome-editing system by forming nanocomplexes is presented. It was found that the PEGylated chitosan-mediated delivery of CRISPR-Cas9 not only protected loaded nucleic acids from the stresses of nebulization, but also enabled mucus penetration and intracellular uptake into the target cells. Compared to nebulizers and propellant-driven metered-dose inhalers (MDIs), dry powder inhalers (DPIs) have several advantages including physical and chemical stability, dose payload, no need for propellant or battery, portability, and ease of actuation. In Chapter 3, thin film freezing (TFF) technique was employed to develop PEGylated chitosan/CRISPR-Cas9 dry powder formulations with different cryoprotective excipients at varying concentrations. Two dry powder formulations with suitable aerodynamic particle size distributions for pulmonary delivery and desired intracellular uptake were refined. In Chapter 4, the feasibility of the aerosolization of mRNA-loaded lipid nanoparticle (LNP-mRNA) and the effects of LNP-mRNA compositions on the properties and potency of aerosolized LNP-mRNA formulations by design of experiments (DOE) was explored. Four LNP-mRNA formulations with relatively high intracellular uptake before and after nebulization were discovered. Two of the four formulations were delivered to mice intratracheally and it was found one formulation showed promising in vivo protein expression after nebulization.Pharmaceutical Science