臺灣博碩士論文加值系統

法布瑞氏症(Fabry disease)為一種罕見的性聯遺傳隱性之溶小體醣脂類儲積症，主要是一種負責製造α -半乳糖甘酵素的基因缺陷，缺陷會造成醣脂質globotriaosylceramide (GL-3) 無法被代謝，這些醣脂質會堆積在心臟、腎臟、皮膚和其他不同組織的溶小體中，造成各種器官的傷害。目前法布瑞氏症的治療方式為酵素替代療法，是透過給予重組人體alpha Gal A (rh-α-GLA)來清除細胞內無法被清除而累積的GL-3。然而，rh-α-GLA用來清除GL3也有其限制，rh-α-GLA在細胞生理中較不穩定，容易迅速地被降解。此外，目前在體外缺乏適當的疾病模式作為提高酵素替代療法治療的醫藥研究。因此，建立一個法布瑞氏症的細胞模式作為篩選有潛力藥物的平台來延長酵素替代療法的效力是有其價值的。為了製造GLA基因的缺陷，我們使用CRISPR / Cas9基因剪輯系統去除HEK293T細胞中GLA基因，使細胞中完全檢測不出內生性GLA的蛋白表現量和酵素活性，作為提供一個空白的背景以檢測rh-α-GLA在細胞中的藥物動力學。而rh-α-GLA在剃除GLA的細胞中會隨著時間延長而降解，其半衰期約為24小時，因此我們想利用rh-α-GLA與有潛力的藥物或小分子共同作用在此一細胞株上，觀察是否可延長GLA的活性。首先，我們發現chaperone N-butyldeoxygalactonojirimycin (NB-DGJ) 和蛋白酶抑制劑E64分別與rh-α-GLA作用在細胞上後，相較於單獨給予rh-α-GLA可以延長rh-α-GLA的活性兩倍之多，除此之外，rh-α-GLA與NB-DGJ或E64合併作用後也可以減少更多的GL-3在法布瑞氏症病人纖維母細胞中的累積量。有鑑於此方法可以有效的鑑定有潛力的藥物是否可以延長rh-α-GLA的效力，於是我們廣泛篩選64個藥物以期找尋到更多可以延長rh-α-GLA活性的藥物，並且發現諸如Calpain inhibitor II、E64C、 2-NBDG、β-D-Galactose pentapivalate、2-Deoxy-D-galactose、Finasteride、Diazepam、 Theophylline、Trazodone、Benzamidine、3-Methyladenine、Carbamazepine、 Selegiline、Sulpiride和Fluorouracil等藥物能有效延長rh-α-GLA在細胞內的活性。總結本研究，經由建立剔除GLA基因的細胞株，能有效的作為體外篩選藥物的平台，進而篩選可延長rh-α-GLA活性的藥物，以避免rh-α-GLA在人體中快速地被降解。

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.

Contents

中文摘要................................ i
Abstract............................... iii
Contents............................... v
List of Figures........................ vii
List of Tables......................... ix
Chapter 1 Introduction..................1
1.1 Fabry disease...................... 2
1.2 Enzyme replacement therapy (ERT)... 4
1.3 CRISPR/Cas system.................. 5
1.4 Chaperone.......................... 8
1.5 Protease inhibitor................. 10
Chapter 2 Aim.......................... 12
Chapter 3 Materials and methods........ 14
3.1 CRISPR genome editing.............. 15
3.2 Fibroblast cultures................ 16
3.3 Cell viability Assay............... 16
3.4 Western blotting................... 17
3.5 GLA enzyme activity................ 18
3.6 Immunofluorescence analysis........ 19
3.7 Enzyme Replacement Therapy (ERT)... 19
3.8 Genomic DNA extraction.................20
3.9 Statistical analysis............... 20
Chapter 4 Results...................... 21
4.1 The GLA deficient cell lines were established via CRISPR/Cas9 system..................... 22
4.2 HEK293T GLA-null cell lines were used as a platform for investigating exogenous rh-α-GLA cellular pharmacokinetics....................... 24
4.3 Co-administration of the pharmacological chaperone NB-DGJ with ERT improved the stability and activity of rhα-GLA in the GLA-null cell line..................... 25
4.4 Co-administration of the protease inhibitor E64 with ERT improved the stability and activity of rhα-GLA in the GLA-null cell line.............................. 28
4.5 NB-DGJ and E64 maintained intracellular amount of rhα-GLA and reduced GL-3 accumulation in Fabry patient-derived fibroblasts............................ 30
4.6 Expand the drug candidates for improving the stability and activity of rhα-GLA in the GLA-null cell line.......... 31
Chapter 5 Discussion................... 33
References............................. 39
Appendices............................. 46


List of Figures

Figure 1. gRNA design for CRISPR/Cas9 system........... 47
Figure 2. GLA activity in selected CRISPR edited HEK293T clones................................................. 48
Figure 3. The sequencing of selected CRISPR edited clones with one nucleotide insertion............................ 49
Figure 4. Cell proliferation of CRISPR edited clones were not affected after genome editing......................... 50
Figure 5. Enzyme activity and protein expression of rhα-GLA in the GLA-null cell lines................................ 51
Figure 6. Enzyme activity and protein expression of rhα-GLA in the GLA-null cell lines................................ 52
Figure 7. rh-α-GLA was decreased with time and had a half-life of 24 hr in the GLA-null cells......................... 53
Figure 8. The appropriate concentrations of chemical chaperones............................................. 54
Figure 9. Co-administration of Fabrazyme and 1-DGJ, NB-DGJ for 5 hr increased the enzyme activity and protein stability of rhα-GLA in vitro....................................... 55
Figure 10. Co-administration of Replagal and 1-DGJ, NB-DGJ for 5 hr increased the enzyme activity and protein stability of rhα-GLA in vitro....................................... 56
Figure 11. Co-administration of Fabrazyme and 1-DGJ, NB-DGJ for 24 hr increased the enzyme activity and protein stability of rhα-GLA in vitro....................................... 57
Figure 12. Co-administration of Replagal and 1-DGJ, NB-DGJ for 24 hr increased the enzyme activity and protein stability of rhα-GLA in vitro....................................... 58
Figure 13. The appropriate concentrations of protease inhibitors............................................. 59
Figure 14. Co-administration of Fabrazyme and E64 for 5 hr increased the enzyme activity and protein stability of rhα-GLA in vitro............................................... 60
Figure 15. Co-administration of Replagal and E64 for 5 hr increased the enzyme activity and protein stability of rhα-GLA in vitro............................................... 61
Figure 16. Co-administration of Fabrazyme and E64 for 24 hr increased the enzyme activity and protein stability of rhα-GLA in vitro............................................... 62
Figure 17. Co-administration of Replagal and E64 for 24 hr increased the enzyme activity and protein stability of rhα-GLA in vitro............................................... 63
Figure 18. Immunostaining of rh-α-Gal A in IVS4+919G>A Fabry patient fibroblasts after co-administration of Fabrazyme and 1-DGJ, NB-DGJ, E64....................................... 64
Figure 19. Immunostaining of rh-α-Gal A in IVS4+919G>A Fabry patient fibroblasts after co-administration of Replagal and 1-DGJ, NB-DGJ, E64....................................... 65
Figure 20. Immunostaining of GL-3 in IVS4+919G>A Fabry patient fibroblasts after co-administration of Fabrazyme and 1-DGJ, NB-DGJ, E64............................................... 66
Figure 21. Immunostaining of GL-3 in IVS4+919G>A Fabry patient fibroblasts after co-administration of Replagal and 1-DGJ, NB-DGJ, E64............................................... 67
Figure 22. The candidates that significantly increased the rh-α-GLA activity after 24 hr co-treatment with Replagal in vitro. ........................................................68

List of Tables

Table 1. List of the drugs for screening in the present study ........................................................72

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