Next Article in Journal
Rat Aquaporin-5 Is pH-Gated Induced by Phosphorylation and Is Implicated in Oxidative Stress
Next Article in Special Issue
Correlations between Endomyocardial Biopsies and Cardiac Manifestations in Taiwanese Patients with the Chinese Hotspot IVS4+919G>A Mutation: Data from the Fabry Outcome Survey
Previous Article in Journal
Estrogenic Endocrine Disrupting Chemicals Influencing NRF1 Regulated Gene Networks in the Development of Complex Human Brain Diseases
Previous Article in Special Issue
The Large Phenotypic Spectrum of Fabry Disease Requires Graduated Diagnosis and Personalized Therapy: A Meta-Analysis Can Help to Differentiate Missense Mutations
Article Menu
Issue 12 (December) cover image

Export Article

Open AccessArticle
Int. J. Mol. Sci. 2016, 17(12), 2089;

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

Institute of Pharmacology, National Yang-Ming University, Taipei 11221, Taiwan
Department of Medical Research, Taipei Veterans General Hospital, Taipei 11217, Taiwan
School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan
Division of Cardiology & Department of Medicine, Taipei Veterans General Hospital, Taipei 11217, Taiwan
Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
Genomics Research Center, Academia Sinica, Taipei 11574, Taiwan
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]   |  


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

Graphical abstract

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

Supplementary material


Share & Cite This Article

MDPI and ACS Style

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.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics



[Return to top]
Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top