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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 胡務亮(Wuh-Liang Hwu) | |
| dc.contributor.author | Mei-Yi Yao | en |
| dc.contributor.author | 姚玫宜 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:08:27Z | - |
| dc.date.available | 2021-11-03 | |
| dc.date.available | 2022-11-24T03:08:27Z | - |
| dc.date.copyright | 2021-11-03 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-10-27 | |
| dc.identifier.citation | 1. Chiang, S.-C., et al., The Timely Needs for Infantile Onset Pompe Disease Newborn Screening—Practice in Taiwan. 2020. 6(2): p. 30. 2. Chien, Y.-H., et al., Pompe disease in infants: improving the prognosis by newborn screening and early treatment. 2009. 124(6): p. e1116-e1125. 3. Chien, Y.-H., et al., Long-term prognosis of patients with infantile-onset Pompe disease diagnosed by newborn screening and treated since birth. 2015. 166(4): p. 985-991. e2. 4. Darrow, J.J.J.D.D.T., Luxturna: FDA documents reveal the value of a costly gene therapy. 2019. 24(4): p. 949-954. 5. Conlon, T.J., et al., Preclinical toxicology and biodistribution studies of recombinant adeno-associated virus 1 human acid α-glucosidase. 2013. 24(3): p. 127-133. 6. Corti, M., et al., Evaluation of readministration of a recombinant adeno-associated virus vector expressing acid alpha-glucosidase in Pompe disease: preclinical to clinical planning. 2015. 26(3): p. 185-193. 7. Lim, J.-A., L. Li, and N.J.F.i.a.n. Raben, Pompe disease: from pathophysiology to therapy and back again. 2014. 6: p. 177. 8. Kohler, L., R. Puertollano, and N.J.N. Raben, Pompe disease: from basic science to therapy. 2018. 15(4): p. 928-942. 9. Taverna, S., et al., Pompe disease: pathogenesis, molecular genetics and diagnosis. 2020. 12(15): p. 15856. 10. Concolino, D., F. Deodato, and R.J.I.J.o.P. Parini, Enzyme replacement therapy: efficacy and limitations. 2018. 44(2): p. 117-126. 11. Fiumara, A. Enzyme replacement therapy (ERT) in pompe disease. in Italian journal of pediatrics. 2014. Springer. 12. Salabarria, S., et al., Advancements in AAV-mediated gene therapy for Pompe disease. 2020. 7(1): p. 15-31. 13. Khanna, R., et al., The pharmacological chaperone AT2220 increases recombinant human acid α-glucosidase uptake and glycogen reduction in a mouse model of Pompe disease. 2012. 7(7): p. e40776. 14. Byrne, B.J., et al., Pompe disease gene therapy. 2011. 20(R1): p. R61-R68. 15. Bak, R.O., N. Gomez-Ospina, and M.H.J.T.i.G. Porteus, Gene editing on center stage. 2018. 34(8): p. 600-611. 16. Ledford, H. and E.J.N. Callaway, PIONEERS OF CRISPR GENE EDITING WIN CHEMISTRY NOBEL. 2020. 586(7829): p. 346-347. 17. Li, H., et al., Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. 2020. 5(1): p. 1-23. 18. FDA. What is Gene Therapy? 2018 07/25/2018 [cited 2018 07/25]; Available from: https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/what-gene-therapy. 19. Gardlík, R., et al., Vectors and delivery systems in gene therapy. 2005. 11(4): p. RA110-RA121. 20. Corti, M., et al., Safety of intradiaphragmatic delivery of adeno-associated virus-mediated alpha-glucosidase (rAAV1-CMV-hGAA) gene therapy in children affected by Pompe disease. 2017. 28(4): p. 208-218. 21. Ronzitti, G., et al., Progress and challenges of gene therapy for Pompe disease. 2019. 7(13). 22. Hordeaux, J., et al., Long-term neurologic and cardiac correction by intrathecal gene therapy in Pompe disease. 2017. 5(1): p. 1-19. 23. Ou, L., et al., ZFN-mediated in vivo genome editing corrects murine hurler syndrome. 2019. 27(1): p. 178-187. 24. Chandrasegaran, S.J.C. and g.t. insights, Recent advances in the use of ZFN-mediated gene editing for human gene therapy. 2017. 3(1): p. 33. 25. Wang, Q., et al., CRISPR-Cas9-mediated in vivo gene integration at the albumin locus recovers hemostasis in neonatal and adult hemophilia B mice. 2020. 18: p. 520-531. 26. Seluanov, A., Z. Mao, and V.J.J. Gorbunova, Analysis of DNA double-strand break (DSB) repair in mammalian cells. 2010(43): p. e2002. 27. Zhang, J.-P., et al., Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. 2017. 18(1): p. 1-18. 28. Persons, D.A.J.M.T., Lentiviral vector gene therapy: effective and safe? 2010. 18(5): p. 861-862. 29. Venditti, C.P.J.N.B., Safety questions for AAV gene therapy. 2021. 39(1): p. 24-26. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80521 | - |
| dc.description.abstract | 龐貝氏症(Pompe disease)是屬於溶小體儲積症，為罕見單基因隱性遺傳疾病。GAA基因的變異會造成acid α-glucosidase缺乏，酵素的缺乏導致肝醣在溶小體內無法被分解成葡萄糖進而堆積，所以也被歸類在肝醣儲積症第二型(Glycogen storage disease type II)。肝醣的堆積會造成許多組織的功能受到影響包括肌肉、心臟甚至是中央神經系統，最後會導致患者因為呼吸衰竭而死亡。 目前對於龐貝氏症的治療，被FDA所許可的只有酵素替代性療法(Enzyme replacement therapy，ERT)。早期ERT的介入的確能提高患者的存活率及活動能力，但由於需終身注射及身體會引發免疫反應對抗注射的酵素，且無法解決根本的問題，所以目前仍有許多不同的治療方法同步在研究中，基因治療也是其中之一。導入完整的GAA序列讓細胞自己去持續的製造所缺乏的acid α-glucosidase進而分解肝醣改善症狀，這就是基因療法的策略。 本篇論文中我們應用CRISPR剪切的專一性及利用細胞同源重組修補的機制將human GAA基因放置於一個安全又穩定的表達區域Acta1 gene中，以達到讓該基因能穩定又安全的在特定區域進行表達，並產生所缺乏的GAA蛋白以利於未來基因療法之開發。此外，本篇論文中也利用在GAA的導入序列5’端與3’端加上sgRNA切點希望轉染時能同時被Cas9切割而呈直線狀態來提高同源重組的效力，期待能將更多的GAA基因置入到Acta1 gene中。我們首先利用軟體選出6組不同的guide RNA轉染老鼠C2C12細胞，再利用細胞分選儀收取帶有GFP的細胞，並用T7E1試驗來找出切割效力最好的sgRNA，利用Cas9使目標位置產生Double-strand break希望產生同源重組的修補機制將human GAA cDNA導入目標位置。 我們在qPCR的試驗中不但可以看到Human GAA的表現量而且在5’端與3’端加上sgRNA切點組別表現量較只有Human GAA的組別有顯著上升(p <0.001)。再透過西方墨點法的蛋白質電泳分析結果中，同樣的可以看到Human GAA 蛋白在5’端與3’端加上sgRNA切點的組別產生的GAA蛋白，較只有human GAA 的組別產生更多。 根據本實驗結果，我們找到能在Acta1 gene上切割效力最好的位點，可以在老鼠C2C12細胞中增加human GAA的表現量。希望能為未來的基因治療提供基礎的研究。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T03:08:27Z (GMT). No. of bitstreams: 1 U0001-2610202122435200.pdf: 2925768 bytes, checksum: 36c3e2230157241a0f79750945867c09 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 誌 謝 i 中文摘要 ii Abstract iv 目 錄 vi 圖目錄 viii 表目錄 ix 第1章 研究背景與動機 1 1.1 前言 1 1.2 龐貝氏症 1 1.2.1 疾病介紹 1 1.2.2 治療方法 2 1.3 基因編輯 3 1.4 基因治療 3 第2章 研究目的與實驗架構 5 2.1 研究目的 5 2.2 實驗架構 6 第3章 研究材料及方法 7 3.1 實驗材料 7 3.2 實驗方法 7 3.2.1 細胞培養(cell culture) 7 3.2.2 質體建構(plasmid construction) 8 3.2.3 DNA轉染(DNA transfection) 8 3.2.4 細胞分選儀(Cell sorting) 9 3.2.5 DNA 萃取 9 3.2.6 T7E1 試驗 9 3.2.7 qPCR 試驗 9 3.2.8 蛋白質電泳及西方墨點法(Western blot analysis) 11 3.2.9 Statistical analysis 12 第4章 實驗結果 13 4.1 Aim1：To select the most effective target site via CRISPR/Cas system - gRNA挑選結果 13 4.2 Aim1：To select the most effective target site via CRISPR/Cas system - gRNA細胞轉染結果 13 4.3 Aim 2：To improvement HR rate - 質體建構 13 4.4 Aim 2：To improvement HR rate - T7E1分析gRNA效力 14 4.5 Aim 2：To improvement HR rate - GAA在C2C12 細胞的表現量 14 4.6 Aim 2：To improvement HR rate - GAA蛋白在C2C12細胞的表現 15 第5章 討論 16 參考文獻 18 | |
| dc.language.iso | zh-TW | |
| dc.subject | 龐貝氏症 | zh_TW |
| dc.subject | 基因編輯 | zh_TW |
| dc.subject | CRISPR | zh_TW |
| dc.subject | 同源重組 | zh_TW |
| dc.subject | Pompe disease | en |
| dc.subject | gene editing | en |
| dc.subject | Homologous recombination | en |
| dc.subject | CRISPR | en |
| dc.title | 開發GAA序列導入之基因治療對抗龐貝氏症 | zh_TW |
| dc.title | Developing a gene therapy strategy for treating Pompe disease by introducing GAA coding sequence | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 簡穎秀(Hsin-Tsai Liu),李妮鍾(Chih-Yang Tseng) | |
| dc.subject.keyword | 龐貝氏症,基因編輯,CRISPR,同源重組, | zh_TW |
| dc.subject.keyword | Pompe disease,gene editing,CRISPR,Homologous recombination, | en |
| dc.relation.page | 33 | |
| dc.identifier.doi | 10.6342/NTU202104280 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-10-28 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
| 顯示於系所單位： | 分子醫學研究所 | |
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