Evaluating a base editing approach for targeting the dominant mutation Y955C in the mitochondrial DNA polymerase

DNA polymerase gamma (Pol-γ) is a heterotrimeric complex responsible for the replication of the mitochondrial genome (mtDNA). It is composed of a large catalytic subunit (Pol-γA), encoded by the nuclear POLG gene, and two smaller accessory subunits (Pol-γβ), encoded by the nuclear POLG2 gene. Mutations in POLG have been recognized among the most prevalent causes of inherited mitochondrial disease, resulting from the impaired function of the enzyme, leading to accumulation of large-scale mtDNA deletion, respiratory chain deficiency and heteroplasmy. Pathogenic variants in POLG have been associated with a spectrum of overlapping phenotypes, with age of onset ranging from infancy to late adulthood, with at least 6 already defined syndromes: Alpers-Huttenlocher Syndrome (AHS), one of the most severe and lethal manifestations typically onsets in infancy, Childhood Myocerebrohepatopathy Spectrum (MCHS) presenting within 3 years of life characterized by severe myopathy and cognitive impairment, Myoclonic Epilepsy Myopathy Sensory Ataxia (MEMSA) presenting in adolescence, defined by cerebellar ataxia and epilepsy, Ataxia Neuropathy Spectrum (ANS) which is a group of related conditions primarily causing neuropathy, ataxia and seizures, autosomal recessive Progressive External Ophthalmoplegia (arPEO) and autosomal dominant Progressive External Ophthalmoplegia (adPEO) both defined by progressive paralysis of the extraocular muscles. Currently, treatment is minimal and mainly centred on management of complications and progression, hence this study provides an insight to a possible permanent solution to an inevitable genetic cage. In my internship, I used cellular models to evaluate a base editing approach to correct the human autosomal dominant Y955C mutation associated with the most common disease genotype currently observed in late-onset conditions such as PEO and Parkinsonism. Specifically, I tested seven engineered single-guide RNAs (sgRNAs) targeting both the human Y955C variant and the murine Y933C analogue, which were combined with two cytosine base editor variants (CBE-NG and CBE-SpRy). HEK cell lines were transfected through lipofection and success of the procedure was verified through fluorescent microscopy. Finally, the editing efficiency was evaluated via Sanger sequencing through quantification of the on target G-to-A conversion within the specified editing window.