科技部新聞稿

基因醫學再突破

全球首創CRISPR-AI雙向基因調控系統並用於再生醫學

日期：108年4月9日

發稿單位：工程技術研究發展司

聯絡人：程弘研究員

電話：2737-7776

E-mail：hcheng@nstc.gov.tw

在科技部計畫長期支持下，清華大學化工系胡育誠講座教授與中國醫藥大學林進裕助理教授及長庚醫院骨科張毓翰醫師組成跨校、跨領域合作團隊，領先世界開發出了新型雙向基因調控系統，並用於調節間葉幹細胞的分化路徑，進而可促進骨組織再生。此成果對再生醫學研究及病人的組織再生將有重大幫助。

人體多種組織如膝關節軟骨及頭蓋骨自我修復能力不佳，若是膝關節軟骨受損便會進一步造成退化性關節炎及病人疼痛。間葉幹細胞可分化成軟骨、硬骨或脂肪等不同細胞，是用於細胞治療，修復組織的重要細胞來源。但實際應用時，幹細胞內有多種調控分化的基因表現不受控制，因此往往無法分化成適當的細胞，造成修復效果不佳。因此如何精準調控基因表現便成了促進組織再生及調節身體機能的重要關鍵。

全球首創CRISPR-AI雙向基因調控系統

為了精準調控基因表現，胡育誠教授團隊以CRISPR/Cas9基因編輯技術為基礎，領先全世界首創CRISPR-AI雙向基因調控系統，可在幹細胞內同時活化與抑制不同基因的表現。團隊利用突變後的Cas9蛋白(dCas9)，並設計兩種導引RNA (sgRNA)骨架。第一種sgRNA骨架(sgRNAa)，可與dCas9及促進基因表現的轉錄因子結合，並標定到促進幹細胞往軟骨細胞分化的基因Sox9。第二種sgRNA骨架(sgRNAi) 可與dCas9及轉錄抑制因子(用於抑制基因表現)結合，並標定到促進脂肪分化的基因PPAR-g。

世界首創應用於組織再生

目前全世界尚未有同時活化及抑制多種基因以促進組織再生之前例。胡育誠教授將此CRISPR-AI系統送入幹細胞後，與原來幹細胞相比，可同時活化Sox9表現17倍，並抑制PPAR-g表現達70%。藉由同時活化(Activation)與抑制(Inhibition)兩種路徑的重要調控基因，此CRISPR-AI 系統可以促進幹細胞往軟骨分化，並且抑制細胞往脂肪分化，因此大幅提升幹細胞分化成軟骨細胞的效率。將此細胞送入頭蓋骨組織缺陷，可以先形成軟骨再分化成硬骨，因此大幅提升頭蓋骨的修復效率。此研究領先全世界，首創開發CRISPR-AI雙向基因調控系統並用於組織再生，已於2019年發表在頂尖國際期刊 核酸研究(Nucleic Acids Research)。

CRISPR-AI可用於細胞療法及癌症治療

胡育誠教授表示，細胞內有複雜的基因網路，因此基因調控是非常重要的。但是要如何雙向調控基因表現仍是一大難題，也還未應用到幹細胞及組織再生。此CRISPR-AI雙向基因調控系統可以同時精準調控多種基因的表現，導引幹細胞分化成特定細胞，未來也可以用於幹細胞研究，並且用於修復關節軟骨，治療退化性關節炎，或修復其他組織。目前癌症治療的新趨勢為應用免疫細胞進行免疫療法，CRISPR-AI系統也具潛力用於改造免疫細胞，增加免疫細胞毒殺癌細胞能力。胡育誠教授正執行科技部台俄合作計畫與俄羅斯團隊進行後續研究。

〈參考資料〉國際期刊發表資料：

1. Truong, V. A., Hsu, M-N., Nguyen, N.T.K., Lin, M-W., Shen, C.-C., Lin, C.-Y., Hu, Y.-C.* 2019. July. CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration.Nucleic Acids Research. 47: e74 (IF 11.147).

     研究成果聯絡人

胡育誠

美國醫學與生物工程學會(AIMBE) 會士

科技部化工學門召集人

清華講座教授

國立清華大學化工系

30013 新竹市光復路二段101號

Tel: 886-3-5718245

Email: ychu@mx.nthu.edu.tw

web:  http://www2.che.nthu.edu.tw/~biohu/

Press Release

April 9, 2020

Breakthrough in gene medicine ―

Taiwan scientists develop CRISPR-AI bi-directional gene regulation system for tissue regeneration

A collaborative team from the National Tsing Hua University, China Medical University and Chang Gung Memorial Hospital in Taiwan has developed, for the first time, a new bi-directional gene regulation system for the control of stem cell differentiation and tissue regeneration.  This work will substantially foster tissue regeneration of patients and benefit stem cell research.

Many tissues in humans, such as articular cartilage and calvarial bone, have limited ability to self-repair. For instance, articular cartilage at the knee degener-ates upon damage, hence losing the ability to provide cushion and resulting in pain at the knee.  Mesenchymal stem cell can differentiate into cartilage, bone or fat cell and is an important cell source for tissue repair by regeneration. During differentiation, however, many essential genes are not well regulated, thus leading to poor cell differentiation and tissue regeneration. Precise manipulation of gene expression is crucial to promote tissue repair.

First CRISPR-AI system for bi-directional gene regulation

To manipulate gene expression, Prof. Yu-Chen Hu at National Tsing Hua University and his collaborators lead the world and develop a novel CRISPR-AI bi-directional gene regulation system to simultaneously activate and inhibit different genes in stem cells.  The team utilizes a dCas9 protein and designs two single guide RNA (sgRNA) scaffold. The first sgRNA binds with dCas9 and transcription factors to target Sox9 gene that promotes stem cell differentiation to cartilage cell. The second sgRNA binds with dCas9 and transcription repressors and targets to PPAR- gene that directs stem cell differentiation to fat cell.

First application of bi-directional gene regulation system in tissue regenera-tion

To date, there is no precedent to promote tissue regeneration by simultaneous activation and inhibition of multiple genes. Prof Yu-Chen Hu and his team show that delivery of the CRISPR-AI system into stem cells can activate Sox9 for 17-fold and repress PPAR- for 70%, when compared with the original stem cell.  By concurrent activation and inhibition of two genes controlling two differentiation paths, the CRISPR-AI system can substantially promote stem cell differentiation towards cartilage and repress differentiation towards fat.  Implantation of the engineered cells into bone defects significantly promotes the tissue regeneration. This study paves a new avenue to developing CRISPR-AI for bi-directional gene regulation and tissue repair, and is published in the top international journal “Nucleic Acids Research” in 2019.

CRISPR-AI holds promise for cell therapy and cancer therapy

Prof. Yu-Chen Hu points out that gene regulation is crucial for cells, but bi-directional and precise control of gene expression remains challenging and has yet to be explored for stem cell and tissue regeneration. This CRISPR-AI system can specifically direct the cell differentiation, thus holding promise for stem cell re-search and for repair of cartilage defects or other tissues. A new trend in cancer therapy is the use of immune cells for immunotherapy. CRISPR-AI is also promising to modulate gene expression in immune cells and enhance their ability to kill cancer cells.   

Reference：

1. Truong, V. A., Hsu, M-N., Nguyen, N.T.K., Lin, M-W., Shen, C.-C., Lin, C.-Y., Hu, Y.-C.* 2019. July. CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration.Nucleic Acids Research. 47: e74 (IF 11.147).

Media Contact

Yu-Chen Hu, PhD

Tsing Hua Chair Professor,

Department of Chemical Engineering,National Tsing Hua University

Tel: 886-3-5718245

Email: ychu@mx.nthu.edu.tw

Hong Cheng

Program Manager

Dept. of Eng. & Technology, MOST

Tel:866-2-7377776

Email:hcheng@nstc.gov.tw