Construction of human artificial chromosome with animal-free chromosome engineering technology

Research Project

Project/Area Number	18K15671
Research Category	Grant-in-Aid for Early-Career Scientists
Allocation Type	Multi-year Fund
Review Section	Basic Section 52050:Embryonic medicine and pediatrics-related
Research Institution	Tokyo University of Pharmacy and Life Science
Principal Investigator	Uno Narumi 東京薬科大学, 生命科学部, 助教 (30733357)
Project Period (FY)	2018-04-01 – 2021-03-31
Project Status	Completed (Fiscal Year 2020)
Budget Amount *help	¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000) Fiscal Year 2020: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000) Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000) Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Keywords	ヒト人工染色体 / 微小核細胞融合法 / ゲノム編集 / 遺伝子導入ベクター / HAC / iPS細胞 / 再生医療 / CRISPR/Cas9 / 巨大染色体領域削除 / ヒト人工染色体ベクター / 染色体導入法 / MMCT
Outline of Final Research Achievements	Human artificial chromosome (HAC) vector is an atractive tool as a novel gene delivery vectors for gene- and cell-therapy. However, there is a risk of contamination with animal-derived proteins or infection with unknown viruses because HAC vectors were constructed by using chicken cells and Chinese hamster cells. In this study, we investigated the development of a novel chromosome engineering technology to eliminate the entire human chromosome gene region and construct HAC vectors in human cells using genome editing technology. In addition, we established a novel chromosome transfer technique for transferring an arbitrary chromosome from human iPS cells to different human cell lines. The results of this research will contribute greatly not only to the clinical application of HAC vectors but also to the research on disease treatment using human iPS cells.
Academic Significance and Societal Importance of the Research Achievements	HACベクターは、搭載する遺伝子サイズに制限がなく、宿主染色体を傷つけずに、独立して保持され、長期間安定な遺伝子発現を示す。このことから、デュシェンヌ型筋ジストロフィー症の根治治療をはじめとして、様々な遺伝子・細胞移植治療への応用が期待されている。しかしながら、作製の際に異種動物細胞を使用していることから、医薬品の製造管理及び品質管理の基準（GMP: Good Manufacturing Practice）に関する省令に適していない。本研究成果である、異種動物細胞不使用のHACベクター運用技術は、HACベクターを用いた新規遺伝子・細胞移植治療の開発に向けた最大の課題を克服するものである。