Development of the New Generation Humanize Bio-Cornea(3/3)-3

View Statistics Email Alert RSS Feed

Information

Details

Project title

Code/計畫編號

MOST106-2622-B001-001-CC1

Translated Name/計畫中文名

新世代擬人化生物人工角膜開發(III)-3

Project Coordinator/計畫主持人

Hong-Yi Gong

Funding Organization/主管機關

National Science and Technology Council

Co-Investigator(s)/共同執行人

吳金洌
耿全福

Department/Unit

Institute of Cellular and Organismic Biology, Academia Sinica

Website

https://www.grb.gov.tw/search/planDetail?id=12202553

Year

2017

Start date/計畫起

01-06-2017

Expected Completion/計畫迄

01-05-2018

Bugetid/研究經費

2500千元

ResearchField/研究領域

畜牧獸醫

目前全球角膜性失明人口約有 1000 萬人，且每年以60 萬人的速度增加中，但現今一年僅有約 10 萬個眼角膜可供移植，眼角膜嚴重缺乏。歐盟一項發表於Science Translational Medicine 的研究指出，利用基因轉殖合成人類膠原蛋白製備之生物合成眼角膜，期待能為等待角膜捐贈病患提供有效替代方案。本計畫合作廠商柏登生醫公司已開發出第一代以吳郭魚鱗片做為人工生物眼角膜之技術，在實驗動物中已經證實可行，但魚鱗中主要的魚類膠原蛋白質，在未來進入人體移植時，可能會產生人體排斥的問題，因此以基因轉殖技術將人類膠原蛋白基因轉入吳郭魚鱗片表現，生產新一代＂擬人化＂魚鱗，實為有效的解決之道，也是廠商急需提早佈局的專利技術之一。擬人化動物模式是將人類的基因、細胞或組織成功轉移至實驗動物，讓實驗動物帶有人類的細胞或表現人類的基因使實驗成果能更有效應用於人類。本計畫之目標在於利用基因轉殖與CRISPR/Cas 9 基因剔除技術，建立只表現人類膠原蛋白1A1 及1A2 之擬人化吳郭魚鱗，生產可做為人工生物眼角膜開發之吳郭魚擬人化魚鱗，完成後，將進行鱗片結構分析及進行進一步動物試驗，未來將技轉給柏登生醫進行臨床測試。在第一年之計畫當中，發現吳郭魚鱗膠原蛋白僅含有第一型，且已成功選殖人類膠原蛋白1A1 及1A2 基因，並完成吳郭魚膠原蛋白1a1、1a2 啟動子及角蛋白4 啟動子之分析，也成功建立吳郭魚膠原蛋白1a1 啟動子啟動人類膠原蛋白1A1 及1A2 之基因轉殖斑馬魚；在第二年計畫中成功建立吳郭魚膠原蛋白啟動子啟動人類膠原蛋白1A1 及1A2 之基因轉殖吳郭魚F0，並利用CRISPR/Cas 9 基因剔除技術，構築針對吳郭魚內生性第一型膠原蛋白1a1a、1a1b 及1a2 基因之guide RNA 載體，體外轉錄後配合 Cas9 mRNA 以顯微注射方式注入吳郭魚胚胎，7 天後以T7E1 assay 檢測全魚DNA 發現膠原蛋白1a1b guide RNA 成功作用在吳郭魚之基因組；在第三年的計畫中，我們將繼續進行人類膠原蛋白1A1 及 1A2 之基因轉殖吳郭魚F1-F2 的建立，並繼續進行吳郭魚內生性col1a1a 與1a2 基因的剔除，分析擬人化基因轉殖吳郭魚鱗片的特性、結構分析，並將擬人化魚鱗交由柏登生醫公司進行動物實驗。It has been estimated that up to 10 million in the world people suffer corneal blindness. Only 100,000 corneal transplants are performed each year worldwide due to the lack of access to donor tissue. A research published in the Science Translational Medicine showed that collagen based biosynthetic corneal implants could offer a safe and effective alternative to the implantation of human tissue to help address the current donor cornea shortage. The Body Organ Biomedical Corporation has developed the first type of bio-cornea from tilapia scales, and transplanted into rat and rabbit, the results are positive, but this may cause immune rejection in human transplantation in future clinical application. Therefore, the company tries to resolve this potential problem. A new generation of “Humanized＂ scale is an effective solution and could be the best product for clinical usage. In this proposal, we will transfer human collagen 1A1 and 1A2 genes into tilapia and knock-out fish collagen 1A1 and 1A2 genes by CRISPR/Cas 9 technology to obtain a humanized tilapia expressing human collagen in the scales for artificial cornea production. At the first year, we analyzed that the tilapia scale only have type I collagen. And we successfully construct the human collagen 1A1 and 1A2 genes. And we already analyzed the promoter assay of tilapia collagen 1A2 and keratin 4. And we established the transgenic zebrafish driven by tilapia collagen 1A1 promoter. Further, we will establish the transgenic tilapia. In second year we successfully establish transgenic human col1a1、1a2 tilapia with green and red fluorescence expression driven by col1a1 2K promoter and confirm the expression of human collagen 1a2 expression in F2 generation transgenic zebrafish. And we also generate col1a1a、col1a1b、 col1a2 guide RNA targeting endogenous tilapia type I collagen genes and confirm that col1a1b guide RNA lead to DNA double strain break by T7E1 assay. In the third year of the program, we will continue to establish the human collagen 1A1 and 1A2 transgenic tilapia F1-F2, and continue to knock-out endogenous col1a1a and 1a2 gene by CRISPR/Cas 9. The characteristics, structure and function of “humanized fish scales＂ will be analyze, and process for animal experiments by the Body Organ Biomedical Corporation (BOBC).

Keyword(s)

人工眼角膜
魚鱗
人類膠原蛋白質1A1/1A2
基因轉殖
基因剔除
Biocornea
fish scale
human collagen 1A1/1A2
transgenic
knock-out

DSpace CRIS

Development of the New Generation Humanize Bio-Cornea(3/3)-3

Details

Description