Using CRISPR/Cas9-Mediated GLA-null Cell Lines as An In Vitro Drug Screening Model for Fabry Disease

• Main Authors: Miao-Chia Yang, 楊苗佳
• Other Authors: Shih-Hwa Chiou
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/49926843078539991979

碩士 === 國立陽明大學 === 藥理學研究所 === 104 === Fabry disease is a hereditary, X-linked lysosomal storage disease resulting from deficient activity of the lysosomal α-galactosidase A. It leads to progressive accumulation of glycosphingolipids particularly globotriaosylceramide (GL-3) in lysosomes of the heart, kidneys, skin and various tissues. Regular administration of recombinant human alpha Gal A (rh-α-GLA), termed enzyme replacement therapy (ERT) is currently available as the only effective treatment for the Fabry patients with GL-3 accumulation. However, the rh-α-GLA driven GL-3 clearance has the limitations, i.e. rh-α-GLA is physiologically instable and quickly degraded in cells. Moreover, lacking of an appropriate in vitro disease model restricted the pharmaceutical studies for improving the ERT treatment. Therefore, it is worth to establish a cell model of Fabry disease (FD) as the platform to screen the potential candidates for prolonging its potency. By utilizing the CRISPR/Cas9 genome editing system, we generated the GLA disruption in HEK293T cells that was completely devoid of detectable GLA protein expression and enzyme activity, providing a clear background to investigate rh-α-GLA cellular pharmacokinetics. The administrated rh-α-GLA was decreased with time and had a half-life of 24 hrs in the GLA-null cells. Base on the GLA deficient cell line, we applied to discover the potential drug or small molecular to restore rh-α-GLA activity. Co-treatment of chaperone drug, N-butyldeoxygalactonojirimycin (NB-DGJ), and protease inhibitor, E64, with ERT significantly prolonged rh-α-GLA activity by over two-folds compared to ERT alone. In addition, NB-DGJ and E64 significantly decreased GL-3 accumulation in the Fabry patients-derived fibroblast. Next, we expanded the screening range of drug and identified the activity for discovering other potential drugs. We screened 64 drugs combining ERT in GLA-null cells and discovered that Calpain inhibitor II, E64C, 2-NBDG, β-D-Galactose pentapivalate, 2-Deoxy-D-galactose, Finasteride, Diazepam, Theophylline, Trazodone, Benzamidine, 3-Methyladenine, Carbamazepine, Selegiline, Sulpiride and Fluorouracil could prolong rh-α-GLA activity. By creating this model, we provide a novel in vitro tool with which to screen potential compounds to avoid short period of GLA activity in human body.