title: Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease.
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 ofGLAin 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.
authors.affiliation: Institute of Pharmacology, National Yang-Ming University, Taipei 11221, Taiwan. shy770307@gmail.com.
authors.forename: Hui-Yung
authors.initials: HY
authors.lastname: Song
authors.affiliation: Department of Medical Research, Taipei Veterans General Hospital, Taipei 11217, Taiwan. skechiang@gmail.com.
authors.forename: Huai-Chih
authors.initials: HC
authors.lastname: Chiang
authors.affiliation: Department of Medical Research, Taipei Veterans General Hospital, Taipei 11217, Taiwan. jan740709@gmail.com.
authors.forename: Wei-Lien
authors.initials: WL
authors.lastname: Tseng
authors.affiliation: Department of Medical Research, Taipei Veterans General Hospital, Taipei 11217, Taiwan. Pingwu821006@gmail.com.
authors.forename: Ping
authors.initials: P
authors.lastname: Wu
authors.affiliation: Department of Medical Research, Taipei Veterans General Hospital, Taipei 11217, Taiwan. cschien6688@gmail.com.
authors.forename: Chian-Shiu
authors.initials: CS
authors.lastname: Chien
authors.affiliation: Department of Medical Research, Taipei Veterans General Hospital, Taipei 11217, Taiwan. hbleu@vghtpe.gov.tw.|School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan. hbleu@vghtpe.gov.tw.|Division of Cardiology & Department of Medicine, Taipei Veterans General Hospital, Taipei 11217, Taiwan. hbleu@vghtpe.gov.tw.
authors.forename: Hsin-Bang
authors.initials: HB
authors.lastname: Leu
authors.affiliation: Department of Medical Research, Taipei Veterans General Hospital, Taipei 11217, Taiwan. molly0103@gmail.com.
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Yang
authors.affiliation: Department of Medical Research, Taipei Veterans General Hospital, Taipei 11217, Taiwan. monglien@gmail.com.
authors.forename: Mong-Lien
authors.initials: ML
authors.lastname: Wang
authors.affiliation: Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. yjjongnctu@gmail.com.
authors.forename: Yuh-Jyh
authors.initials: YJ
authors.lastname: Jong
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 11574, Taiwan. winschen@gate.sinica.edu.tw.
authors.forename: Chung-Hsuan
authors.initials: CH
authors.lastname: Chen
authors.affiliation: School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan. wcyu@vghtpe.gov.tw.|Division of Cardiology & Department of Medicine, Taipei Veterans General Hospital, Taipei 11217, Taiwan. wcyu@vghtpe.gov.tw.
authors.forename: Wen-Chung
authors.initials: WC
authors.lastname: Yu
authors.affiliation: Institute of Pharmacology, National Yang-Ming University, Taipei 11221, Taiwan. shchiou@vghtpe.gov.tw.|Department of Medical Research, Taipei Veterans General Hospital, Taipei 11217, Taiwan. shchiou@vghtpe.gov.tw.|School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan. shchiou@vghtpe.gov.tw.|Institute of Clinical Medicine, National Yang-Ming University, Taipei 11221, Taiwan. shchiou@vghtpe.gov.tw.
authors.forename: Shih-Hwa
authors.initials: SH
authors.lastname: Chiou
mesh_terms: D015295:Antigens, Tumor-Associated, Carbohydrate / Q000378:metabolism; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D016923:Cell Death / Q000187:drug effects; D004353:Drug Evaluation, Preclinical; D004795:Enzyme Stability / Q000187:drug effects; D000795:Fabry Disease / Q000188:drug therapy*; D005347:Fibroblasts / Q000378:metabolism; D000072669:Gene Editing; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D007976:Leupeptins / Q000008:administration & dosage / Q000494:pharmacology; D008954:Models, Biological; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D000519:alpha-Galactosidase / Q000378:metabolism*
chemical_list: D015295:Antigens, Tumor-Associated, Carbohydrate; C472766:Gb3 antigen; D007976:Leupeptins; D011994:Recombinant Proteins; D000519:alpha-Galactosidase; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: CRISPR; Fabry disease; MG132; drug screening; enzyme replacement therapy (ERT)