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Int. J. Mol. Sci. 2016, 17(12), 2089; doi:10.3390/ijms17122089

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

1
Institute of Pharmacology, National Yang-Ming University, Taipei 11221, Taiwan
2
Department of Medical Research, Taipei Veterans General Hospital, Taipei 11217, Taiwan
3
School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan
4
Division of Cardiology & Department of Medicine, Taipei Veterans General Hospital, Taipei 11217, Taiwan
5
Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
6
Genomics Research Center, Academia Sinica, Taipei 11574, Taiwan
7
Institute of Clinical Medicine, National Yang-Ming University, Taipei 11221, Taiwan
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Academic Editor: Ritva Tikkanen
Received: 20 September 2016 / Revised: 30 November 2016 / Accepted: 5 December 2016 / Published: 13 December 2016
View Full-Text   |   Download PDF [2356 KB, uploaded 13 December 2016]   |  

Abstract

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 α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-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 rhα-GLA cellular pharmacokinetics. The half-life of administrated rhα-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rhα-GLA significantly restored the GLA enzyme activity by two-fold compared with rhα-GLA alone. Furthermore, co-treatment of rhα-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rhα-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 rhα-GLA as well as for screening candidates to prolong rhα-GLA potency. Using this model, we demonstrated that MG132 prolongs rhα-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment. View Full-Text
Keywords: Fabry disease; CRISPR; enzyme replacement therapy (ERT); drug screening; MG132 Fabry disease; CRISPR; enzyme replacement therapy (ERT); drug screening; MG132
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Song, H.-Y.; Chiang, H.-C.; Tseng, W.-L.; Wu, P.; Chien, C.-S.; Leu, H.-B.; Yang, Y.-P.; Wang, M.-L.; Jong, Y.-J.; Chen, C.-H.; Yu, W.-C.; Chiou, S.-H. Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease. Int. J. Mol. Sci. 2016, 17, 2089.

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