Evaluation of CRISPR/nCas9 for gene editing on mucopolysaccharidosis type IIIB

Mucopolysaccharidosis IIIB (MPS IIIB) is a lysosomal disease (LD) characterized by the deficiency of the alpha-N-acetylglucosaminidase (NAGLU) enzyme caused by mutations in the gene encoding for it. The deficiency of this lysosomal enzyme generates the accumulation of glycosaminoglycans (GAGs), especially heparan sulfate (HS), in the lysosomes of the cell as well as extracellularly, which causes different clinical conditions mainly affecting the central nervous system (CNS). To date, there is no approved therapy for the treatment of MPS IIIB, however, gene editing with CRISPR/nCas9 and non-viral vectors as a gene delivery mechanism arises as a treatment model alternative that has shown encouraging results in other types of LDs such as GM2 gangliosidoses and MPS IVA. In this sense, in the present study the use of the genomic editing tool CRISPR/nCas9 for the of an expression cassette with NAGLU cDNA at the AAVS1 locus of the human genome was evaluated for the first time in an in vitro model. The results showed a successful recombination and construction of a donor vector with NAGLU cDNA, and an increase in long-term enzyme activity (30 days) reaching values of 71% of normal NAGLU activity. Additionally, a decrease in the average lysosomal mass was observed within 30 days of treatment, but not in total GAGs. In addition, we evaluated a polymeric non-viral vector, which showed enhanced transfection efficiency compared to lipofectamine, and may represent a novel tool for the design of gene therapy strategies. These results serve as a basis for future experiments that contribute to the actual knowledge about the benefits, advantages and risks of using CRISPR/nCas9 as a new gene therapy alternative for MPS IIIB.