Patient-specific Fabry disease cell models as a tool to evaluate the amenability to chaperone therapy

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by the deficient activity of the lysosomal enzyme α-galactosidase A (GLA), leading to intracellular globotriaosylceramide (Gb3) accumulation. Patients with amenable mutations can be treated with a pharmacological chaperone (migalastat) to restore endogenous GLA activity. Current tests to determine patients’ amenability are limited to heterologous mutation expression in HEK cells. We established a new cell culture model based on immortalized urine cells from Fabry patients, as well as CRISPR-CAS 9-mediated HEK cells lacking endogenous GLA activity. The model was used to test amenability of GLA mutations p.N215S and p.L294S as well as IVS2+1 A>T nonsense mutation, serving as negative control. Characterization of the cell lines was performed by real-time PCR and fluorescence microscopy. Amenability to chaperone treatment was quantified by enzyme activity assays and intracellular Gb3 measurements using flow cytometry and compared to patients’ biochemical response. Under chaperone treatment (>13 months), patients with the p.N215S mutation (n=5) showed a significant reduction of plasma lyso-Gb3 (p<0.05). By contrast, lyso-Gb3 levels in carriers of the p.L294S Mutation (n=3) increased under treatment (p<0.05). While the amenability assay in wildtype HEK293T cells suggested amenability for both mutations (p<0.05), chaperone-treatment provoked increased GLA activity (p<0.0001) and intracellular Gb3 reduction (p<0.05) only in immortalized p.N215S cells but not in p.L294S or IVS2+1 A>T cells. Amenabilities were confirmed using GLA knock-out cells for amenability assays. In vitro GLA activity assays predicted p.L294S as an amenable mutation for chaperone therapy. Our study with patient-specific immortalized cells revealed that the amenability of p.L294S might be too low, which was confirmed by the poor biochemical response to migalastat observed in patients with p.L294S. Additional studies are required to further analyze mutations with endogenous GLA activity <5% of wildtype for their amenability to chaperone treatment in a patient-specific Setting.