Genome editing on GM2 gangliosidoses fibroblasts using CRISPR/nCas9

GM2 gangliosidoses is a group of lysosomal diseases (LDs) characterized by mutations on the genes encoding for the α- or β-subunits of β-hexosaminidases A (αβ) and B (ββ). These mutations lead to Tay-Sachs and Sandhoff diseases, respectively; in which an abnormal lysosomal storage of GM2 ganglioside is observed. Consequently, a neurodegenerative decline occurs in patients suffering the disease with occasional visceromegaly. Currently, some CRISPR/Cas9-based gene therapy approaches have been reported to induce the knock-in of the HexM, a chimeric protein composed by the α subunit, the stable homodimer interface of β-subunits and the β-subunit domains predicted to interact with GM2AP, into the albumin locus showing interesting results. Despite the above, the chimeric proteins could be like neoantigens that the human immune system could recognize. In this scenario, we decided to evaluate the therapeutic potential of the CRISPR/nCas9-mediated insertion of α- and β-subunits using in vitro models of Tay-Sachs (GM00515) and Sandhoff (GM00317) diseases in the AAVS1 locus as safe-harbor. Upon the lipofection of the donor plasmid and CRISPR/nCas9 system, we reached up to 11% and 13.1% of the wild-type HexA and HexB activity, respectively, 30 days post-transfection. In contrast to Tay-Sachs cells, Sandhoff cells showed a time-dependent enzyme activity increase in the culture media (~53% of wild type at day 30) suggesting that HexB could be secreted to the extracellular space and mediate cross-correction. In concordance with this, lysosomal mass and mitochondria-dependent ROS was decreased in both models supporting the previous findings. In conclusion, we demonstrated that knock-in of the human α- and β-subunits could be a promising therapeutic alternative for GM2 gangliosidoses and we postulate that our approach could reduce the potential immune response derived of chimeric proteins like HexM.