為初代人類自然殺手細胞建立CRISPR 基因編輯平台

CRISPR genome engineering platform for primary human natural killer cells

賴敏碁(Min-Chi Lai)

指導教授 ： 凌嘉鴻

國立臺灣大學/生命科學院/生化科學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202101268

摘要

自然殺手細胞是在癌症免疫治療中非常有潛力的先天性免疫細胞。為了研究和增強自然殺手細胞的功能，強大且完整的基因編輯工具是必須的。但是，自然殺手細胞對於傳統的基因編輯方式例如質體轉染和病毒轉導都是很困難的，所以為了克服這些限制，我在這篇論文中為了自然殺手細胞建立一個強大的CRISPR基因編輯的平台並且是利用電轉染組裝好之Cas9: guide RNA核蛋白送入細胞並進行基因剔除。為了要送入大片段的轉殖基因到自然殺手細胞中，我們選擇了擁有不會嵌入宿主基因體和大轉殖基因乘載量(38kb)的桿狀病毒，這能讓我們送入許多不同的基因。然而我們發現野生型的桿狀病毒病無法感染自然殺手細胞。在這篇論文當中，我觀察並編輯了自然殺手細胞表面受體使其能被野生型的桿狀病毒感染，我也利用了細胞內先天免疫反應的抑制藥物，希望能避免桿狀病毒感染細胞後產生的免疫反應。這篇論文當中的發現提供了桿狀病毒感染自然殺手細胞後重要的現象，也為未來以桿狀病毒為基礎的自然殺手細胞基因編輯平台奠定了重要的基礎。

關鍵字

自然殺手細胞 ； CRISPR ； 電轉染 ； 桿狀病毒 ； 免疫療法

並列摘要

Natural killer (NK) cells are potent innate immune cells that show promising efficacy in cancer immunotherapy. To study and enhance NK cell functions, robust genetic tools are needed. However, NK cells are challenging to modify genetically by conventional methods such as plasmid transfection and viral transduction. To overcome this limitation, we established a robust CRISPR genome editing platform for NK cells by the nucleofection of pre-assembled Cas9 ribonucleoproteins (RNP). Our platform is versatile to enable efficient gene knock-out. To facilitate transgene delivery in NK cells, baculovirus is an attractive vector because of its non-integrative nature and large transgene capacity (38 kb), which is sufficient for many gene cargos. Unfortunately, the standard baculoviral vector cannot transduce NK cells without modifications. In this thesis, I summarize my effort to manipulate the NK cells surface receptors to enable baculoviral transduction and to prevent intracellular immune responses in the baculoviral transduced NK cells. The findings of this study provide important insights into the intracellular anti-viral defense mechanisms in NK cells, and establish the foundation to develop a robust baculoviral transduction platform for NK cell engineering.

並列關鍵字

Natural killer cell ； CRISPR ； Nucleofection ； Baculovirus ； Immunotherapy

參考文獻

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