Graham, Carina; (2024) Preliminary Exploration of a DNA Editing Strategy to Treat Cystic Fibrosis using Homology-Independent Targeted Integration and Receptor-Targeted Nanoparticles. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cystic fibrosis (CF) is a common life-limiting disorder. Dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) protein hinders mucociliary clearance in the airways, leading to a cycle of infection, inflammation, and tissue damage. Pharmaceutical intervention is available for patients of the most common CF-causing genotype (F508del), but burden of care remains high, and patients carrying non-F508del variants are ineligible for drug use. CRISPR-Cas9 is a promising technology to eliminate underlying causes of genetic disorders. We hypothesised firstly that a CRISPR approach based in homology independent targeted integration could be used to generate a “one size fits all” treatment strategy for CF patients harbouring “undruggable” genotypes ineligible for pharmaceutical intervention, and secondly that receptor-targeting nanoparticles (RTNs) could be used to deliver CRISPR machinery to differentiated airway epithelial cells. We characterised a CF mutant cell line homozygous for an undruggable variant in order to provide a phenotypic baseline for future treatment studies. Characterisation via Ussing chamber and scanning ion conductance microscopy confirmed that variant cells exhibited lack of ion transport, dehydrated airway surface liquid, and decreased mucosal elasticity. To provide proof of concept for a CRISPR-based strategy using homology-independent targeted integration (HITI), CRISPR-Cas9 machinery and donor were transfected via plasmids and used to edit an eGFP reporter construct into the AAVS1 safe harbour locus. The reporter construct inserted with an efficiency of ~40%. RTNs were then investigated for their ability to transfect airway epithelial cells; to enhance editing efficiency, RTNs were packaged with CRISPR ribonucleoprotein (RNP). RTNs were assayed for their biophysical characteristics (size and charge) and their ability to transfect airway epithelial cells in vitro using luciferase-based reporter assays. RTNs’ ability to penetrate human airway mucus was investigated in vitro, establishing that CF mucus provides a significant barrier to RTN mobility in fluid. When transfection of differentiated air-liquid interface (ALI) cultures was attempted, we found negligible transfection using RTNs. These results lay the groundwork for future study. The editing efficiency achieved using HITI exceeds the predicted rate required for rescue of CF pathology; therefore, replacing the reporter construct donor with a CFTR cDNA donor could potentially represent a variant-agnostic treatment for CF. RTNs’ promising performance in submerged cells and isolated non-CF mucus, but not ALI-differentiated non-CF cells, provides insight into the barriers to in vivo RTN-mediated transfection. Future work with these RTNs will require further optimisation of their formulation (addition of polyethylene glycol to lipid formulations and repeat of phage display in ALI to identify new targeting peptides) and ALI transfection protocols (applications of mucolytics, chemicals to loosen tight junctions and increase accessible membrane surface area, paralysis of cilia).

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