臺灣博碩士論文加值系統

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。

Pompe disease is a lysosomal storage disease and a rare monogenic inherited disorder. The mutation in the acid alpha-glucosidase (GAA) can lead to the deficient of an acid alpha-glucosidase and cause a build-up of glycogen in lysosome. Pompe disease is also known as glycogen storage disease type II. It affects many tissues including muscle, heart, and CNS and patients die mainly due to respiratory failure.
Currently Enzyme replacement therapy (ERT) is the only treatment approved by FDA. Early treatment with ERT can maintain muscle function and improve patients’ survival rate; however, the treatment needs to be frequently readministered and may cause an immune response against the injected enzyme. It can’t solve the underlying problem. To address these limitations, there are some experimental treatments for Pompe disease in various stages of development. Gene therapy is one of them. Introducing human GAA coding sequence and produce the missing enzyme to reduce glycogen by cell itself is the strategy of gene therapy.
In this article we insert human GAA coding sequence in the safe and specific Acta1 locus via CRISPR-Cas9 leading to sustained enzyme expression and is conducive to future gene therapy development. We also optimize donor templet by adding 5’ and 3’ sgRNA cutting site to enhance the homologous recombination rate. The donor plasmid contains a gRNA cutting site and can be linearized when co-transfected with CRISPR-Cas9.We hope it can enhance homologous recombination rate and insert more human GAA in Acta1 locus. First, we select the six different gRNA by CHOPCHOP and find the most efficient one by T7E1. Generation of a double-strand break in mouse Acta1 locus is via CRISPR-Cas9 and insert the human GAA cDNA by homologous recombination (HDR)
We found that the Human GAA expression in qPCR analysis and the level in adding 5’ and 3’ sgRNA cutting site group is higher than only human GAA donor (p <0.001). Human GAA protein was observed by western blot. Similar Western analysis showing human GAA protein level in adding 5’ and 3’ sgRNA cutting site group is higher than only human GAA donor.
The result show that we can target the Acta1 locus via the most efficient gRNA and enhance human GAA in mouse C2C12 cell line. We hope the result provides basic research for Pompe disease gene therapy.

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

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