Gene Correction on a Roll

Abstract

Gene-editing technologies offer a promising perspective for the treatment of rare metabolic disorders through targeted correction of genetic mutations. In this thesis, we focus on the development and application of prime editing as a precision technique for gene therapy. In Chapter 2, we developed prime editing conditions for the correction of pathogenic mutations in patient-derived organoids. Genetic correction led to functional restoration of enzyme activity without detectable off-target effects, as demonstrated by whole genome sequencing. In Chapter 3, we introduced fluoPEER, a fluorescence-based screening system for rapid optimization of prime editing. We showed that prime editing efficiency depends on the cell cycle, with increased efficiency during the S phase. Chapter 4 describes the use of DdCBE, a mitochondria-targeted base editor, which we used to both induce and correct pathogenic mutations in mitochondrial DNA. In Chapter 5, we analyzed liver models and developed HLCompR, a bioinformatics tool that compares transcriptomics to assess the suitability of liver models. We demonstrated that existing models lack key liver functions. Therefore, in Chapter 6, we developed a new liver organoid model, HeLLO, that better mimics essential liver functions. In Chapter 7, we elaborated a business case for the use of HeLLO in liver toxicity prediction. In Chapter 8, we investigated delivery methods for prime editing, including virus-like particles and lipid nanoparticles. Finally, in Chapter 9, we outlined the requirements for clinical implementation, including regulatory aspects, GMP production, and a sustainable funding model.