Development of genome editing technology with low off-target risk and high efficiency in human pluripotent stem cells

• Project/Area Number: 15K06924
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: System genome science
• Research Institution: Keio University
• Principal Investigator: SONE Takefumi 慶應義塾大学, 医学部(信濃町), 訪問研究員 (00379091)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: ゲノム編集 / iPS細胞 / CRISPR / ノックイン / TALEN / 遺伝子治療 / 相同組換え / 疾患研究 / ウイルスベクター

Outline of Final Research Achievements

Genome editing technology has made it familiar for us to knock-out a gene, but it is still difficult to knock-in a certain DNA sequence precisely into a genomic locus. In this study, a simple and effective method was developed to construct a gene knock-in vector with a drug-resistant gene cassette. Using the vector in combination with TALEN of CRISPR expression vectors, responsible genes of iPSC lines derived from various disease patients were successfully corrected or those of control iPSC lines were modified to have mutation. Especially for CRISPR expression vector, an all-in-one construct was developed to express two sets of sgRNAs for two dual-nickase Cas9 pairs to cut two genomic loci at the same time for lower the risk of off-target effect. Combination of this vector with two knock-in vectors with two different drug-resistant gene cassette, a reliable homo knock-in system was also developed and confirmed for its feasibility.

Research Products

• [Int'l Joint Research] Johns Hopkins University(米国)
• [Journal Article] Generation of D1-1 TALEN isogenic control cell line from Dravet syndrome patient iPSCs using TALEN-mediated editing of the SCN1A gene (2018)
  • Author(s): Tanaka Yasuyoshi、Sone Takefumi、Higurashi Norimichi、Sakuma Tetsushi、Suzuki Sadafumi、Ishikawa Mitsuru、Yamamoto Takashi、Mitsui Jun、Tsuji Hitomi、Okano Hideyuki、Hirose Shinichi
  • Journal Title: Stem Cell Research Volume: 28 Pages: 100-104
  • DOI: 10.1016/j.scr.2018.01.036
  • Peer Reviewed / Open Access
• [Journal Article] Down-regulation of ghrelin receptors on dopaminergic neurons in the substantia nigra contributes to Parkinson's disease-like motor dysfunction. (2018)
  • Author(s): Suda Y, Kuzumaki N, Sone T, Narita M, Tanaka K, Hamada Y, Iwasawa C, Shibasaki M, Maekawa A, Matsuo M, Akamatsu W, Hattori N, Okano H, Narita M.
  • Journal Title: Mol Brain. Volume: 11 Issue: 1 Pages: 6-6
  • DOI: 10.1186/s13041-018-0349-8
  • Peer Reviewed / Open Access
• [Journal Article] Efficient induction of dopaminergic neuron differentiation from induced pluripotent stem cells reveals impaired mitophagy in PARK2 neurons. (2017)
  • Author(s): Suzuki S, Akamatsu W, Kisa F, Sone T, Ishikawa KI, Kuzumaki N, Katayama H, Miyawaki A, Hattori N, Okano H.
  • Journal Title: Biochem Biophys Res Commun. Volume: 489 Issue: 1 Pages: 88-93
  • DOI: 10.1016/j.bbrc.2016.12.188
  • Peer Reviewed / Open Access
• [Journal Article] Cochlear Cell Modeling Using Disease-Specific iPSCs Unveils a Degenerative Phenotype and Suggests Treatments for Congenital Progressive Hearing Loss (2017)
  • Author(s): Makoto Hosoya, Masato Fujioka, Takefumi Sone, Satoshi Okamoto, Wado Akamatsu, Hideki Ukai, Hiroki R. Ueda, Kaoru Ogawa, Tatsuo Matsunaga, Hideyuki Okano
  • Journal Title: Cell Reports Volume: 18 Issue: 1 Pages: 68-81
  • DOI: 10.1016/j.celrep.2016.12.020
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] 2. iPS細胞技術とゲノム編集技術を駆使した筋萎縮性側索硬化症病態モデルの創成と治療への展望 (2016)
  • Author(s): 曽根岳史、一柳直希、藤森康希、岡野栄之
  • Journal Title: 実験医学(増刊) Volume: 34 Pages: 146-157
• [Journal Article] Establishment of in vitro FUS-associated familial amyotrophic lateral sclerosis model using human induced pluripotent stem cells. (2016)
  • Author(s): Naoki Ichiyanagi, Koki Fujimori, Masato Yano, Chikako Ishihara-Fujisaki, Takefumi Sone, Tetsuya Akiyama, Yohei Okada, Wado Akamatsu, Takuya Matsumoto, Mitsuru Ishikawa, Yoshinori Nishimoto, Yasuharu Ishihara, Tetsushi Sakuma, Takashi Yamamoto, Hitomi Tsuiji, Naoki Suzuki, Hitoshi Warita, Masashi Aoki, Hideyuki Okano
  • Journal Title: Stem Cell Reports Volume: 6 Issue: 4 Pages: 496-510
  • DOI: 10.1016/j.stemcr.2016.02.011
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Controlling the regional identity of hPSC-derived neurons to uncover neuronal subtype specificity of neurological disease phenotypes. (2015)
  • Author(s): Kent Imaizumi, Takefumi Sone, Keiji Ibata, Koki Fujimori, Michisuke Yuzaki, Wado Akamatsu, Hideyuki Okano
  • Journal Title: Stem Cell Reports Volume: 5 Issue: 6 Pages: 1010-1022
  • DOI: 10.1016/j.stemcr.2015.10.005
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] iPS細胞におけるAngelman症候群原因遺伝子UBE3Aノックアウトの作製と神経細胞の表現型探索 (2017)
  • Author(s): 大河内 遼太郎, 曽根 岳史, 石川 充, 奥野 博庸, 中武 悠樹, 洪 実, 小崎 健次郎, 岡野 栄之
  • Organizer: 第16回日本再生医療学会総会
  • Place of Presentation: 仙台国際センター, 仙台
  • Year and Date: 2017-03-07
• [Presentation] 遺伝性パーキンソン病患者由来iPS細胞におけるゲノム編集iPS細胞の樹立 (2017)
  • Author(s): 太田 悦朗, 曽根 岳史, 大日方 友理, 鵜飼 英樹, 久松 知子, 北川 季子, 石川 充, 駒野 肇, 上田泰己, 小幡 文弥, 岡野 栄之
  • Organizer: 第16回日本再生医療学会総会
  • Place of Presentation: 仙台国際センター, 仙台
  • Year and Date: 2017-03-07
• [Presentation] Functional analysis of synaptic gene expression and morphologies in Angelman syndrome patient-derived neurons (2017)
  • Author(s): Mitsuru Ishikawa, Ryotaro Okochi, Hironobu Okuno, Shoma Tanaka, Takefumi Sone
  • Organizer: CDB Symposium 2017
  • Int'l Joint Research
• [Presentation] Generation of gene-corrected iPSC from patient-derived iPSC with familial Parkinson's disease (2017)
  • Author(s): Etsuro Ohta, Takefumi Sone, Yuri Obinata, Hideki Ukai, Tomoko Hisamatsu, Tokiko Kitagawa, Mitsuru Ishikawa, Hajime Komano, Hiroki R. Ueda, Fumiya Obata, Hideyuki Okano
  • Organizer: ISSCR 2017 Annual Meeting
  • Int'l Joint Research
• [Presentation] Multisite Gatewayクローニング技術に立脚した汎用ヒトiPS細胞ゲノム編集ツールボックス (2017)
  • Author(s): 曽根 岳史, 吉松 祥, 塩澤 誠司, 大河内 遼太郎, 石川 充, 木下 実結, 藤森 康希, 川嶋 志保子, 大多 茂樹, 鐘ヶ江 裕美, 三好 浩之, 岡野 栄之
  • Organizer: 日本ゲノム編集学会第2回大会
• [Presentation] Pathophysiological analysis of skeletal muscles in spinal bulbar muscular atrophy (SBMA) by genome editing of CAG repeats. (2017)
  • Author(s): Satoshi Tanaka, Takuji Ito, Akinobu Ota, Takefumi Sone, Daisuke Shimojo, Shiro Imagama, Yoshitaka Hosokawa, Manabu Doyu, Hideyuki Okano, Yohei Okada
  • Organizer: Neuroscience 2017 (SfN’s 47th Annual Meeting)
  • Int'l Joint Research
• [Presentation] ゲノム編集によるポリグルタミン病の病態解析と治療開発の可能性 (2016)
  • Author(s): 田中 智史, 伊藤 卓治, 太田 明伸, 曽根 岳史, 下門 大祐, 小野寺 一成, 今釜 史郎, 細川 好孝, 道勇 学, 勝野 雅央, 祖父江 元, 岡野 栄之, 岡田 洋平
  • Organizer: 第39回分子生物学会年会
  • Place of Presentation: パシフィコ横浜, 横浜
  • Year and Date: 2016-11-30
• [Presentation] mt-Keimaを用いたパーキンソン病iPS細胞におけるマイトファジー解析システムの構築 (2016)
  • Author(s): 鈴木 禎史*, 赤松 和土, 木佐 文彦, 曽根 岳史, 石川 景一, 服部 信孝, 岡野 栄之
  • Organizer: 第38回日本生物学的精神医学会・第59回日本神経化学会大会合同年会
  • Place of Presentation: 福岡国際会議場, 福岡
  • Year and Date: 2016-09-08
• [Presentation] ゲノム編集による筋委縮性側索硬化症(ALS)の病態モデル細胞の創成と遺伝子治療 (2016)
  • Author(s): 曽根 岳史*, 一柳 直希, 藤森 康希, 岡野 栄之
  • Organizer: 日本ゲノム編集学会第1回大会
  • Place of Presentation: 広島国際会議場, 広島
  • Year and Date: 2016-09-06
• [Presentation] ゲノム編集によるポリグルタミン病の病態解析と治療開発の可能性 (2016)
  • Author(s): 田中 智史, 伊藤 卓治, 太田 明伸, 曽根 岳史, 今釜 史郎, 細川 好孝, 道勇 学, 岡野 栄之, 岡田 洋平
  • Organizer: 日本ゲノム編集学会第1回大会
  • Place of Presentation: 広島国際会議場, 広島
  • Year and Date: 2016-09-06
• [Presentation] 遺伝性パーキンソン病患者由来iPS細胞におけるゲノム編集iPS細胞の樹立 (2016)
  • Author(s): 大日方 友理*, 太田 悦朗, 曽根 岳史, 鵜飼 英樹, 久松 知子, 石川 充, 駒野 肇, 上田 泰己, 小幡 文弥, 岡野 栄之
  • Organizer: 第11回日本臨床検査教育学会学術大会
  • Place of Presentation: 神戸常盤大学・神戸国際展示場, 神戸
  • Year and Date: 2016-08-31
• [Presentation] 遺伝性パーキンソン病患者由来iPS細胞のゲノム編集および病態解析 (2016)
  • Author(s): 太田 悦朗*, 曽根 岳史, 大日方 友理, 鵜飼 英樹, 久松 知子, 北川 季子, 石川 充, 駒野 肇, 上田 泰己, 小幡 文弥, 岡野 栄之
  • Organizer: 第39回日本神経科学大会
  • Place of Presentation: パシフィコ横浜, 横浜
  • Year and Date: 2016-07-20
• [Presentation] Establishment of in vitro FUS-associated familial amyotrophic lateral sclerosis model using human induced pluripotent stem cells (2016)
  • Author(s): Naoki Ichiyanagi*, Koki Fujimori, Masato Yano, Chikako Ishihara-Fujisaki, Takefumi Sone, Tetsuya Akiyama, Yohei Okada, Wado Akamatsu, Takuya Matsumoto, Mitsuru Ishikawa, Yoshinori Nishimoto, Yasuharu Ishihara, Tetsushi Sakuma, Takashi Yamamoto, Hitomi Tsuiji, Naoki Suzuki, Hitoshi Warita, Masashi Aoki, Hideyuki Okano
  • Organizer: ISSCR 2016 Annual Meeting
  • Place of Presentation: San Francisco, CA
  • Year and Date: 2016-06-22
  • Int'l Joint Research
• [Presentation] Generation of specific neuronal subtypes from human pluripotent stem cells (2016)
  • Author(s): Kent Imaizumi*, Takefumi Sone, Wado Akamatsu, Hideyuki Okano
  • Organizer: ISSCR 2016 Annual Meeting
  • Place of Presentation: San Francisco, CA
  • Year and Date: 2016-06-22
  • Int'l Joint Research
• [Presentation] ゲノム編集した患者由来疾患iPS細胞の全ゲノム解析によって明らかになったこと (2015)
  • Author(s): 曽根 岳史*、朴 聖俊、田中 泰圭、太田 悦朗、日暮 憲道、中井 謙太、廣瀬 伸一、岡野 栄之
  • Organizer: BMB2015 （第38回日本分子生物学会年会、第88回日本生化学会大会 合同大会）
  • Place of Presentation: 神戸ポートアイランド (兵庫県神戸市)
  • Year and Date: 2015-12-01
  • Invited
• [Presentation] TALEN-mediated genome editing of patient-derived iPSCs for disease model studies. (2015)
  • Author(s): Takefumi Sone*, Yasuyoshi Tanaka, Etsuro Ohta, Makoto Hosoya、Naoki Ichiyanagi, Tetsuya Akiyama, Norimichi Higurashi, Shinichi Hirose, Hideyuki Okano
  • Organizer: TGE2015: Conference on Transposition and Genome Engineering 2015
  • Place of Presentation: 奈良春日野国際フォーラム 甍～I・RA・KA～ (奈良県奈良市)
  • Year and Date: 2015-11-17
  • Int'l Joint Research
• [Presentation] A mutated PENDRIN formed intracellular aggregations in the inner ear cells induced from Pendred syndrome patients-specific iPS cells. (2015)
  • Author(s): 細谷 誠*, 藤岡 正人, 渡部 孝太郎, 岡本 理志, 曽根 岳史, 松永 達雄, 赤松 和土, 小川 郁, 岡野 栄之
  • Organizer: 第38回日本神経科学大会
  • Place of Presentation: 神戸国際会議場・神戸国際展示場 (兵庫県神戸市)
  • Year and Date: 2015-07-28
• [Presentation] PENDRED症候群疾患特異的iPS細胞由来内耳細胞を用いた薬剤スクリーニング (2015)
  • Author(s): 細谷 誠*, 藤岡 正人, 岡本 理志, 曽根 岳史, 松永 達雄, 赤松 和土, 小川 郁, 岡野 栄之
  • Organizer: 第36回日本炎症・再生医学会
  • Place of Presentation: 虎ノ門ヒルズフォーラム (東京都港区)
  • Year and Date: 2015-07-21
• [Presentation] Genome editing of SCN1A in induced pluripotent stem cells to study the pathomechanisms of Dravet syndrome. (2015)
  • Author(s): Yasuyoshi Tanaka*, Taku Uchida, Takefumi Sone, Norimichi Higurashi, Mitsuru Ishikawa, Hideyuki Okano, Shinichi Hirose
  • Organizer: The 13th Asian and Oceanian Congress of Child Neurology
  • Place of Presentation: 台北 (台湾)
  • Year and Date: 2015-05-14
  • Int'l Joint Research
• [Remarks] ドラベ症候群iPS細胞モデルを用いた創薬ハイスループットスクリーニングシステムの確立に向けた基盤研究
  • URL: https://fukuoka-u.repo.nii.ac.jp/?action=repository_uri&item_id=3996&file_id=22&file_no=1
• [Remarks] iPS 細胞を用いて「内耳変性」という難聴の新たな原因と、 その治療薬候補物質を発見
  • URL: https://www.keio.ac.jp/ja/press-releases/files/2017/1/11/170111-1.pdf
• [Remarks] iPS 細胞とゲノム編集技術を用いて 筋萎縮性側索硬化症（ALS）の病態モデルを構築
  • URL: http://www.keio.ac.jp/ja/press_release/2015/osa3qr000001g2lb-att/20160318_01.pdf