Efficient phenotype recovery of MPS IVA fibroblasts after CRISPR/nCas9-mediated genome editing

CRISPR/Cas9 has opened new horizons in the treatment of the genetic diseases and recently it has been tested on several lysosomal storage diseases (LSDs) such as MPS I and GM2 gangliosidoses. Despite its promising advantages, at date this approach has not been tested for the MPS IVA; an LSD in which the loss of GALNS activity leads the lysosomal accumulation of the glycosaminoglycans keratan sulfate and chondroitin-6-sulfate. This accumulation activates several pathological cascades such as oxidative stress and may affect the autophagic-lysosomal system. In this study, we performed a CRISPR/Cas9-mediated knock in of GALNS gene to evaluated in-vitro the recovery of the MPS IVA phenotype. The AAVS1 locus was chosen as the safe harbor for the knock in of a GALNS expression cassette using a nickase of Cas9 and two sgRNA. The nCas9 activity, cassette expression, homologous recombination and Off-target effect was evaluated on HEK293FT cells. A cut efficiency of nCas9 of 36.8% was achieved and non-Off-target was identified using Sanger sequencing. On the other hand, the GM00593 cell line was used as model of MPS IVA to test our system using a lipofection approach. Upon the transfection, we found an increase in the GALNS activity up to 26,8% of wild-type levels 30 days post-transfection. This increase was consistent with a reduction in the lysosomal mass quantified by flow cytometry using LysoTracker Deep Red. Likewise, at this point a slight reduction on the mitochondria-derived ROS was observed upon the staining with MitoSox Red. In conclusion, our results suggest strongly that knock in of GALNS encoding sequence using CRISPR/nCas9 allows the recovery of phenotype in MPS IVA and could be a promising therapeutic alternative for the treatment and/or cure of this LSD.