Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease

The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human alpha Gal A (rh alpha-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rh alpha-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy. Therefore, it is worth establishing a large-expanded in vitro FD model for screening potential candidates, which can enhance and prolong ERT potency. Using CRISPR/Cas9-mediated gene knockout of GLA in HEK-293T cells, we generated GLA-null cells to investigate rh alpha-GLA cellular pharmacokinetics. The half-life of administrated rh alpha-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rh ff -GLA significantly restored the GLA enzyme activity by two-fold compared with rh alpha-GLA alone. Furthermore, co-treatment of rh alpha-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rh alpha-GLA treatment alone. Collectively, the CRISPR/Cas9-mediated GLA-knockout HEK-293T cells provide an in vitro FD model for evaluating the intracellular pharmacokinetics of the rh alpha-GLA as well as for screening candidates to prolong rh alpha-GLA potency. Using this model, we demonstrated that MG132 prolongs rh alpha-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment.