Generation and characterization of a GALC knock-out human oligodendrocyte cell line (MO3.13) using CRISPR/Cas9 methodology for studying Krabbe disease.

Krabbe leukodystrophy (KD) is a rare autosomal recessive disease caused by the defective gene GALC encoding galactosylceramidase, which is an important enzyme in lysosomal catabolism. In physiological conditions, GALC hydrolyzes the galactose ester bonds of glycolipids such as galactosylceramide and galactosylsphingosine (commonly referred to as psychosine, PSY). On the other hand, the GALC activity deficiency leads to PSY accumulation in myelinating cells – oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system – and induces widespread demyelination followed by devasting neurodegeneration. In the scientific literature, it is known that GALC correction and PSY clearance might not be sufficient to completely restore the KD phenotype, suggesting that not yet identified molecular mechanisms could be involved in the KD pathogenesis.

Here, we report on a new in vitro cellular model of Krabbe disease, based on the human oligodendrocyte cell line MO3.13. We took advantage of the genome editing CRISPR/Cas9 system for creating a stable GALC-deficient cell line. The experimental flow was based on several steps for reagent design, construction of single guide RNA (sgRNA)-expressing plasmids, transfection and cell line expansion and characterization. We could successfully isolate and identify an isogenic cell line with a homozygous deletion of 19 nucleotides in exon 7 of the GALC gene. Exon 7 contains the nucleophilic domain, common for all isoforms and important for GALC function. To verify whether this mutation could have altered GALC activity, we performed the quantification of galactosylceramidase activity on cellular lysates by 6-hexadecanoylamino-4-methylumbelliferyl-b-D-galactopyranoside (HMU-βGal) assay. The GALC-/- cells showed a decreased enzymatic activity compared to wild-type ones. These preliminary results suggest the successful generation of a stable model, but further experiments are ongoing to validate this line as a new tool useful to explore the hallmarks of Krabbe disease and to test new interventions against oligodendrocyte alterations.