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

Mucopolysaccharidosis IVA (MPS 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

CRISPR/Cas9-based gene therapy for MPS IVA, recovering up to 40 % of normal GALNS activity,

leading to a positive outcome on classical MPS IVA biomarkers. In this study, we extended our

previous findings by performing 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 in

GM00593 and GM01361 human MPS IVA fibroblasts using a lipofection-based delivery. After 30

days post-transfection with CRISPR/Cas9 systems in the 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 GM01361 and

GM00593 cells, 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 in treated

GM01361 cells. None of these cells transfected with CRISPR/Cas9 plasmids expressing E4orf6 or

E1B showed improvement for the test variables. Together, our results support using CRISPR/Cas9 as a

potential GT-based strategy for treating MPS IVA and highlight the importance of seeking approaches

to improve the impact of gene editing as a therapeutic approach.