Advancing Prime Editing Strategies for the Genetic Correction of Methylmalonic Acidemia Mutations: From Engineered Cell Models to Primary Mouse Fibroblasts

Abstract

Methylmalonic acidemia (MMA) is a rare but severe inherited metabolic disorder caused by mutations in the methylmalonyl-CoA mutase (MUT) gene, for which no curative treatments currently exist. Prime editing has recently emerged as a promising genome-editing technology that enables precise correction of disease-causing mutations without inducing double-stranded DNA breaks, as required by conventional CRISPR/Cas9 approaches. In this thesis, both prime editing and twin prime editing strategies were evaluated to correct two clinically relevant MUT mutations, p.M700K and p.R727X, across various cellular models of MMA. It was first attempted to replicate established twin prime editing experiments from Anzalone et al. at the HEK3 locus and PAH exon 7 in HEK293T cells but substantial challenges in reproducibility were encountered. Subsequently, it was aimed to correct the mouse p.M698K mutation, which mirrors the human p.M700K variant, in engineered HEK293T cells and primary mouse fibroblasts harbouring the mutation. While twin prime editing achieved efficient correction in HEK293T cells, editing outcomes were influenced by the cell line's partial deficiency in mismatch repair (MMR). Editing efficiencies in primary fibroblasts were more variable and challenging to replicate, likely reflecting differences in cellular context and technical barriers. For the p.R727X mutation, a fluoPEER screen was employed to systematically evaluate multiple prime editing guide designs, leading to the identification of a candidate pegRNA with improved editing activity. Overall, our findings emphasize the need to optimize both editing strategies and experimental conditions, as well as to select appropriate cellular models when developing prime editing approaches for the treatment of MMA and similar genetic diseases.