CRISPR gene editing for Cystic Fibrosis: targeting the G542X mutation with base editor engineered virus-like particles
Cystic Fibrosis (CF) is a severe genetic disorder affecting hundreds of thousands of individuals worldwide. Approximately 10% of CF patients currently lack treatment options beyond symptomatic care and face a reduced life expectancy. CRISPR-based gene editing offers the potential for a cure, by addressing the underlying cause of the disease. The doctoral work presented in this dissertation explored the avenue of CRISPR therapeutic gene editing for CF, while also testing and characterizing a recently emerged delivery technology in the form of virus-like particles. This thesis opens with a systematic review of state-of-the-art CRISPR editing strategies and their application to CF-causing variants, highlighting challenges and gaps that guided the subsequent experimental work. Building on these insights, a base editing approach was developed to target G542X - the most common CF-causing mutation ineligible to modulator therapy - using engineered virus-like particles (eVLPs). This strategy successfully rescued the mutation in patient-derived intestinal organoids, and prompted further investigation into the eVLPs. Two titration assays and a luminescence-based reporter system were established to standardize and monitor particle-mediated transduction. Finally, extending the G542X-editing strategy to CF airway cells revealed previously unreported, non-canonical base editing outcomes specific to eVLP delivery, that are further explored in this dissertation. The review, assays and experimental findings presented and discussed in this thesis aim to advance gene editing for CF. It is hoped this work will encourage and support future research to prioritize editing strategies for modulator-unresponsive variants, incorporating rigorous validation of delivery vehicles in relevant cell types, and integrating transcriptomic, functional and in-vivo studies.
Cystic Fibrosis, Gene editing, CRISPR, Virus-like particles
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