Polymer-Based Gene Therapy for Lung Cystic Fibrosis Disease

Lung cystic fibrosis (CF) is a lethal inherited disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, leading to a dysfunctional CFTR protein. Gene therapy offers promise for the treatment of lung CF. However, the two big challenges that have impeded its success are the absence of: (1) an efficient and safe gene delivery reagent to transfect the target cells, and (2) a stable plasmid that enables high and sustained CF transmembrane conductance regulator (CFTR) expression. Thus, the aim of this work is to develop a non-viral CFTR gene replacement system for lung CF disease to overcome these two challenges. Chapter 2 introduce a CpG-depleted CFTR plasmid, incorporating a codon-optimized CFTR sequence driven by the human elongation factor 1α (hEFIα) promoter. This plasmid enhances and sustains CFTR protein expression in lung CF epithelial cells. Chapter 3 explains the development of a novel polymer-based gene replacement treatment approach for treating lung CF disease. A nanoparticle, formed by the selected highly branched PAE (HPAE) with a CpG-depleted CFTR plasmid, demonstrates superior CFTR gene expression and biocompatibility in comparison to commercial gene transfection reagents. The newly developed gene therapy system successfully restored functional CFTR protein production in lung CF epithelial monolayers. Chapter 4 focuses on a scalable concentration method for HPAE/DNA polyplexes using ultrafiltration. This results in a 24-fold increase in concentration without compromising transfection efficiency, showcasing potential for lung delivery applications. Chapter 5 evaluates the nebulised HPAE/DNA polyplexes in vitro and in vivo. While in vitro results demonstrate high shear resistance, further improvements are needed for in vivo transfection efficacy. Chapter 6 provides a comprehensive summary of the entire project, discussing limitations and proposing future directions.