Evaluation of the CRISPR/Cas9 system as genome editing platform for the Mucopolysaccharidosis IV A using a strategy for induction of higher homologous recombination frequency

Mucopolysaccharidosis IVA is a lysosomal storage disease caused by mutations in the gene encoding for the hydrolase N-acetylgalactose-6-sulfate sulfatase (GALNS). GALNS deficiency leads to a progressive buildup of partially degraded chondroitin-6-sulfate and keratan-6-sulfate within the lysosomes. Several therapeutic strategies are under evaluation. Recently, we validated the use of a CRISPR/Cas9 nickase-based GT for MPS IVA, recovering up to 40% of normal GALNS activity, which had a positive outcome on classical MPS IVA biomarkers. In this study, we extended our previous findings by using the co-delivery of the CRISPR/Cas9 system with the proteins E4orf6 or E1B, which are inhibitors of the non-homologous end-joining repair pathway that may favor an increase in the homology-directed repair mechanism. The CRISPR/Cas9 vectors were transfected on GM00593 and GM01361 human MPS IVA fibroblasts by using a lipofection-based delivery. After 30 days post-transfection with CRISPR/Cas9 systems in absence of the E4orf6 or E1B, we found an increase in the GALNS activity up to 4.17 and 11.2-fold when compared to untreated controls, for GM01361 and GM00593 respectively. Partial recovery of the lysosomal mass was achieved in human MPS IVA fibroblasts after treatment, while a modest reduction in oxidative stress was observed only for GM01361. Non-improvement on those parameters was detected when CRISPR/Cas9 plasmids expressing E4orf6 or E1B were transfected. Together, our results support the use of CRISPR/Cas9 as a potential GT-based strategy for the treatment of MPS IVA and highlight the importance of continuing to seek strategies to improve the impact of gene editing as a therapeutic approach.