CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors

Int J Mol Sci. 2022 Sep 14;23(18):10672. doi: 10.3390/ijms231810672.


The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the β-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the β-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the β-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the β-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.

Keywords: CRISPR/nCas9; GM2 gangliosidoses; Sandhoff; Tay–Sachs; genome editing; non-viral vectors.

MeSH terms

  • Deoxyribonuclease I / metabolism
  • Fibroblasts / metabolism
  • G(M2) Activator Protein
  • G(M2) Ganglioside / genetics
  • G(M2) Ganglioside / metabolism
  • Gangliosidoses, GM2* / genetics
  • Gangliosidoses, GM2* / metabolism
  • Gangliosidoses, GM2* / therapy
  • Gene Editing
  • Globosides / metabolism
  • Glycosaminoglycans / metabolism
  • Hexosaminidase A / metabolism
  • Humans
  • Lipopolysaccharides / metabolism
  • Liposomes / metabolism
  • Tay-Sachs Disease* / genetics
  • Tay-Sachs Disease* / metabolism
  • Tay-Sachs Disease* / therapy
  • beta-N-Acetylhexosaminidases / metabolism


  • G(M2) Activator Protein
  • Globosides
  • Glycosaminoglycans
  • Lipopolysaccharides
  • Liposomes
  • G(M2) Ganglioside
  • Deoxyribonuclease I
  • Hexosaminidase A
  • beta-N-Acetylhexosaminidases