Design and application of prime editing to target ARSA P426L mutation in a metachromatic leukodystrophy patient

Abstract

Metachromatic leukodystrophy (MLD) is a rare inherited lysosomal storage disease caused primarily by mutations in the Arylsulfatase A (ARSA) gene. Loss of functional ARSA leads to sulfatide accumulation, causing demyelination in the peripheral and central nervous system. MLD presents in different clinical forms, which all eventually result in severe neurological decline and death. Currently available treatment options are limited. Enzyme replacement therapy (ERT) and allogeneic hematopoietic stem cell transplantation (allo-HSCT) may slow disease progression but do not offer a cure. Libmeldy was recently approved by the European Medicines Agency (EMA) as the first gene therapy for early-onset forms of MLD. However, the potential long-term risk of insertional mutagenesis remains a concern and requires ongoing evaluation. In this project we targeted the ARSA P426L mutation, one of the most common MLD-causing mutations in Europe. We used prime editing, a CRISPR-Cas9 based mechanism, which allows to introduce precise changes into the genome. The experiments were carried out in K562 cells and hematopoietic stem and progenitor cells (HSPCs). If the ARSA P426L mutation could be corrected ex vivo in patient-derived HSPCs prior to transplantation, this approach could offer a potentially curative treatment for patients who are currently lacking effective therapeutic options. Such a strategy would eliminate the need to find a matching donor for allogeneic HSCT, evade its side effects and avoid the risk of insertional mutagenesis associated with Libmeldy. Instead, it would restore physiological ARSA expression by precisely correcting the mutation at its endogenous locus. In K562 cells, we successfully introduced the ARSA P426L mutation using prime editing, achieving mean editing efficiencies of 1.88% with PE2 and 10.20% with PE3. However, no editing was detected in HSPCs of healthy donors. As a result, this study did not progress to target patient-derived HSPCs. Further investigations are needed to focus on optimizing the prime editing mechanism as well as related components in order to achieve higher editing efficiency. Also research about target site specific limitations for prime editing should be closely monitored and taken into account when planning for future projects. Although editing of the ARSA P426L locus was not successful, the high efficiency to induce the IL2RG c.458T>C mutation in HSPCs, which was used as a proof of concept, supports the general feasibility of prime editing based gene therapy approaches.