Genome editing in mucopolysaccharidosis type IVA fibroblasts using CRISPR/Cas9

Mucopolysaccharidosis type IVA (MPS IVA; Morquio A, OMIM 253000) is a monogenic recessive lysosomal disease caused by mutations in the gene encoding for N-acetyl-galactosamine-6-sulfate sulfatase (GALNS). The absence of an active enzyme causes a progressive accumulation of the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin-6-sulfate (C6S) mainly in bone, cartilage, and connective tissue. GAGs build up leads to skeletal dysplasia, organomegaly, respiratory problems and heart valve disease among other symptoms. Currently, there are two different strategies as treatment options for MPS IVA with clinical approval: Enzymatic replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT). Although these strategies have proved promissory results, they still lack effectiveness, especially in reaching bone tissue. Also, ERT does not offer a long-term solution and in the case of HSCT, there is a considerable risk associated with this procedure. Gene therapy offers a potential long-term solution for genetic diseases. Here we explored the potential of the CRISPR/Cas9 system in a gene therapy scenario for MPS IVA targeting the AAVS1 locus for insertion of a functional copy of the GALNS gene under the expression of a CMV promoter. MPS IVA fibroblast were treated with CRISPR/Cas9 system and GALNS donor vector. We confirmed the gene editing in the AAVS1 locus, as well as the reduction in lysosomal mass and the increase in the intracellular GALNS activity after 30 days post-treatment with CRISPR/Cas9 system and GALNS donor vector. This study shows the first evidence about the use of CRISPR/Cas9 on MPS IVA, and will shed light in the design of a novel therapeutic strategy for this disorder.