合成生物技術應用於基因載體設計及細胞調控

Application of synthetic biology in vector design and cell engineering

林美薇(Mei-Wei Lin)

指導教授 ： 胡育誠

國立清華大學/工學院/化學工程學系/博士(2020年)

摘要

合成生物學是一種將基因工程應用在生物系統的方法及工具，從基因體組成、調控、訊息傳遞延伸到細胞設計甚至是整個生物系統的改造。在這個研究裡，我們將合成生物技術應用於兩類生物系統-癌症治療基因載體的設計以及抗體生產細胞的改造。在第一部份-以合成開關控制的桿狀病毒載體用以調控外源基因表達及選擇性毒殺肝癌細胞: 桿狀病毒所發展的抗癌基因載體對於肝癌的治療有相當好的潛力，但應用到生物體內時，常無法避免抗癌基因也在正常細胞表現並產生毒性，為了提高安全性，我們設計了能夠感應細胞特有miRNA圖譜的miRNA感應器，並與RNA結合蛋白-L7Ae組裝成外源基因轉譯的開關，裝載到桿狀病毒載體，研究結果顯示重組的桿狀病毒載體能有效地進入肝細胞和正常細胞，但外源基因僅會在肝癌細胞中的啟動，當使用促凋亡的hBax作為外源基因時，桿狀病毒載體能在肝癌細胞和正常細胞共培養時具選擇性殺死肝癌細胞，這些結果證明了帶有合成開關元件的桿狀病毒載體，能辨識癌細胞執行專一性毒殺。在第二部分-利用CRISPR/Cas13系統進行CHO細胞株多基因調控增加去岩藻糖抗體的產量及活性: 中國倉鼠卵巢 (CHO)細胞是最廣泛使用在生產生物藥的細胞工廠，我們利用CRISPR-Cas13d RNA靶向系統進行CHO細胞系統的調控編輯，我們證實CRISPR-Cas13d有效地抑制LDHA, GFT, DDIT3以及CLU等基因，並造成功能上的改變，包含: 乳酸的產生、岩藻糖醣基化程度、氧化自由基的含量及細胞團塊的形成。再者，結合Sleeping Beauty system使CRISPR-Cas13d模組更穩定在CHO細胞株達到多重基因同時調控並證明整體抗體產量顯著提高，且降低抗體岩藻糖醣基化促使抗體依賴性細胞毒殺效果提升。展現Cas13d在CHO細胞系細胞工程中的應用潛能。綜合上述結果，在生物系統的架構裡，透過裝載合成的外源基因組件，我們優化病毒載體的效能及安全性應用於肝癌治療，並且編輯CHO細胞轉錄體增加抗體活性及產量。

關鍵字

桿狀病毒 ； 載體 ； 凋亡 ； 抗體 ； 去岩藻糖醣基化

並列摘要

Synthetic biology is a tool of applying genetic engineering to biological systems, which comprises genome manipulation, regulation, signal transduction, cell engineering or programming of entire biological systems. In this study, we applied synthetic biotechnology to cancer therapeutic vector design and engineering of antibody-producing cells. Part I-Synthetic switch-based baculovirus for transgene expression control and selective killing of hepatocellular carcinoma cells: Baculovirus (BV) holds promise as a vector for anticancer gene delivery to combat hepatocellular carcinoma (HCC). However, in vivo BV administration inevitably results in BV entry into non-HCC normal cells and leaky anticancer gene expression. To improve the safety, we employed a miRNA sensor and assembled it with RNA binding protein, L7Ae, for BV design. Our result showed the recombinant BV efficiently entered HCC and normal cells, enabled switching ON transgene exclusively in HCC cells. Using pro-apoptotic hBax as the transgene, the switch-based BV selectively killed HCC cells in mixed culture of HCC and normal cells. These data demonstrate the potential of synthetic switch-based BV to distinguish HCC and normal cells for selective killing of HCC cells. Part II-Enhancing the yield and activity of defucosylated antibody produced from CHO-K1 cells using Cas13-mediated multiplex gene targetin: Chinese hamster ovary (CHO) cells are the most widely used in biopharmaceutical cell factories. We have demonstrated CRISPR-Cas13d effectively modulate the expression of several endogenous gene such as LDHA, GFT, DDIT3 and CLU, which also gave rise to the inhibition of lactic acid production, fucosylation, the reactive oxygen species generation, and cell aggregation. Furthermore, exploitation of Sleeping Beauty system-mediated integration of CRISPR-Cas13d steadily suppressed multiplex gene expression in CHO cell and endow the cell with high productivity and trimming of antibody fucosylation relevant to the enhancement of antibody-dependent cell-mediated Cytotoxicity. Collectively, we already implanted the synthetic device into the vector or organism construction for optimizing the effectiveness of viral vectors in liver cancer treatment and engineering the CHO cell transcriptome to increase antibody yield and activity.

並列關鍵字

Baculovirus ； vector ； apoptosis ； antibody ； defucosylated

參考文獻

1. T. Tian et al., MiR-146a and miR-196a-2 polymorphisms are associated with hepatitis virus-related hepatocellular cancer risk: a meta-analysis. Aging (Albany NY) 9, 381-392 (2017).

2. C. L. Chen et al., Suppression of hepatocellular carcinoma by baculovirus-mediated expression of long non-coding RNA PTENP1 and MicroRNA regulation. Biomaterials 44, 71-81 (2015).

3. K. J. Airenne et al., Baculovirus: an insect-derived vector for diverse gene transfer applications. Mol Ther 21, 739-749 (2013).

4. J. Zeng, J. Du, Y. Zhao, N. Palanisamy, S. Wang, Baculoviral vector-mediated transient and stable transgene expression in human embryonic stem cells. Stem cells 25, 1055-1061 (2007).

5. W. Liu et al., MicroRNA-16 targets amyloid precursor protein to potentially modulate Alzheimer's-associated pathogenesis in SAMP8 mice. Neurobiology of aging 33, 522-534 (2012).