iPS cell-based modeling of inherited arrhythmia disorders using the latest genome editing technology

2019 Fiscal Year Research-status Report

• Project/Area Number: 19K17595
• Research Institution: Kyoto University
• Principal Investigator: 烏日揚海 伊敏 京都大学, 医学研究科, 研究員 (10824956)
• Project Period (FY): 2019-04-01 – 2021-03-31
• Keywords: 遺伝子不整脈 / iPS細胞 / 突然死 / ラミン関連心筋症 / 疾患モデル / ゲノム編集 / QT延長症候群

Outline of Annual Research Achievements

本研究では、ゲノム編集技術を用いて作製した致死性家族性不整脈疾患ヒトiPS細胞モデルを用いた発症機序解明、治療法開発を目指した研究を行った。
心臓ナトリウムチャネルとリアノジン受容体遺伝子のcompound hetero異常による重症不整脈症例において、CRISPR-Cas9システムを用いた一塩基置換を行い一遺伝子変異ずつcorrectionしたiPS細胞株の作製に成功した。引き続き分化心筋細胞の機能解析を進行中である。
LMNA遺伝子は、核膜の裏打ち蛋白であるlamin A,Cをコードし、その遺伝子異常によりlaminopathyと呼ばれる様々な疾患を引き起こす。心臓では、拡張型心筋症と心臓伝導障害を呈することが知られている。我々は健常人由来iPS細胞モデルにおいてLMNA遺伝子ノックアウトした株を樹立し、胚様体形成法、monolayer心筋分化法にて心筋分化を行った。ノックアウト株の心筋細胞の拍動数が有意に減少し、ラミン心筋症の表現型の一つを再現できた。パッチクランプ法を用いた電気生理学的解析行ったところ、心臓Naチャネル電流に関しては、健常人由来コントロール心筋細胞と比較し有意な差は認めず、ウエスタンブロッティング法による蛋白解析では、L型カルシウムチャネルの減少がみられた。引き続き疾患表現型の解析を進めている。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

Compound変異を持つ患者由来iPS細胞を樹立し、ゲノム編集技術により変異を一つずつcorrectionした株の作製に成功した。また、LMNA関連心筋症に関してはiPS細胞を用いた疾患モデル構築、発症メカニズムの解明を行っており、進捗状況は概ね順調と考える。

Strategy for Future Research Activity

引き続き遺伝性不整脈、心筋症において疾患特異的iPS細胞モデルの確立、治療法開発を目指した研究を行っていく。Compound変異疾患iPS細胞モデルについては、分化心筋細胞において、組織学的解析（免疫染色、電顕観察）、分子生物学的解析（RNA、蛋白定量）、電気生理学的解析（活動電位や各イオンチャネル電流記録）を行う。表現型確認後、候補治療薬（β遮断薬、Naチャネル阻害薬、Caチャネル拮抗薬など）の薬効評価を進める。LMNA関連心筋症iPS細胞モデルについて、引き続き組織学的解析、分子生物学的解析、電気生理学的機能解析を行い、疾患モデルとしての確立を目指す。治療候補薬スクリーニングに関しては、京都大学医学研究支援センタードラッグデイスカバリセンターを利用する予定。

Causes of Carryover

現在樹立した疾患特異的iPS細胞を用いて次年度で引き続き解析を行うため。iPS細胞培養、心筋分化試薬等にも使用予定。

Research Products

• [Journal Article] Phenotype-Based High-Throughput Classification of Long QT Syndrome Subtypes Using Human Induced Pluripotent Stem Cells (2019)
  • Author(s): Yoshinaga Daisuke、Baba Shiro、Makiyama Takeru、Shibata Hirofumi、Hirata Takuya、Akagi Kentaro、Matsuda Koichi、Kohjitani Hirohiko、Wuriyanghai Yimin、Umeda Katsutsugu、Yamamoto Yuta、Conklin Bruce R.、Horie Minoru、Takita Junko、Heike Toshio
  • Journal Title: Stem Cell Reports Volume: 13 Pages: 394-404
  • DOI: 10.1016/j.stemcr.2019.06.007
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Expertise ヒトiPS細胞由来心筋細胞の電気生理学 (特集 心臓電気生理を理解して不整脈診療に活かす) iPS cell-derived cardiomyocytes as a platform for research on inherited arrhythmias (2019)
  • Author(s): 堀江 稔,Wuriyanghai Yimin,牧山 武
  • Journal Title: Heart view Volume: 23 Pages: 578-585
• [Presentation] Modeling Overlap Syndrome Of Long QT And Brugada Syndrome Associated With CACNA1C-E1115K Which Alters Ion Selectivity With Induced Pluripotent Stem Cells (2019)
  • Author(s): Asami Kashiwa, Takeru Makiyama, Hirohiko Kohjitani, Yimin Wuriyanghai, Yuta Yamamoto, Gao Jungshan, Hai Huang, Taisuke Ishikawa, Seiko Ohno, Futoshi Toyoda, , Seiichi Sato, Minoru Horie , Naomasa Makita, Takeshi Kimura.
  • Organizer: 40th Heart Rhythm Scientific Sessions
  • Int'l Joint Research
• [Presentation] Automatic Computational Action Potential Analyzing System Enables Accurate Drug Response Interpretation and Prediction in Human Induced Pluripotent Stem Cell-derived Cardiomyocytes (2019)
  • Author(s): Hirohiko Kohjitani, Shigeya Kouda, Yukiko Himeno, Takeru Makiyama, Asami Kashiwa, Yimin Wuriyanghai, Yuta Yamamoto, Minoru Horie, Takeshi Kimura, Akinori Noma, Akira Amano.
  • Organizer: 40th Heart Rhythm Scientific Sessions
  • Int'l Joint Research
• [Presentation] Structural Insights into Catecholaminergic Polymorphic Ventricular Tachycardia-associated RyR2 Mutant Channels using a Three-dimensional In Silico Model. (2019)
  • Author(s): J. Gao, T. Makiyama, S. Ohno, Y. Yamamoto, Y. Wuriyanghai, H. Kohjitani, A. Kashiwa, H. Huang, K. Katou, M. Horie, T. Kimura.
  • Organizer: European Society of Cardiology Congress 2019
  • Int'l Joint Research
• [Presentation] KCNQ1 Channel Dysfunction in human induced pluripotent stem cell-derived cardiomyocytes carrying a CALM2-N98S mutation associated with long QT syndrome (2019)
  • Author(s): Yuta Yamamoto, Takeru Makiyama, Yimin Wuriyanghai, Hirohiko Kohjitani, Sayako Hirose, Jingshan Gao, Asami Kashiwa, Huang Hai, Takanori Aizawa, Tomohiko Imamura, Taisuke Ishikawa, Seiko Ohno, Kazuhisa Chonabayashi, Hideki Motomura, Yoshinori Yoshida, Minoru Horie, Naomasa Makita, Takeshi Kimura
  • Organizer: American Heart Association Scientific Sessions 2019
  • Int'l Joint Research
• [Presentation] Morphological and electrophysiological characterization of homozygous lamin A/C knockout human induced-pluripotent stem cell-derived cardiomyocytes. American Heart Association Scientific Sessions 2019 (2019)
  • Author(s): Yimin Wuriyanghai, Takeru Makiyama, Yuta Yamamoto, Hirohiko Kohjitani, Sayako Hirose, Jingshan Gao, Asami Kashiwa, Hai Huang, Takanori Aizawa, Tomohiko Imamura, Seiko Ohno, Minoru Horie, Takeshi Kimura
  • Organizer: American Heart Association Scientific Sessions 2019
  • Int'l Joint Research
• [Presentation] Response of KCNQ1 to Adrenergic Stimulation was Impaired in Human Induced Pluripotent Stem Cell-derived Cardiomyocytes Carrying a CALM2-N98S Mutation Associated with Long QT Syndrome. (2019)
  • Author(s): Yuta Yamamoto, Takeru Makiyama, Yimin Wuriyanghai, Hirohiko Kohjitani, Sayako Hirose, Jingshan Gao, Asami Kashiwa, Huang Hai, Takanori Aizawa, Tomohiko Imamura, Taisuke Ishikawa, Seiko Ohno, Kazuhisa Chonabayashi, Hideki Motomura, Yoshinori Yoshida, Minoru Horie, Naomasa Makita, Takeshi Kimura
  • Organizer: 12th Asia Pacific Heart Rhythm Society Scientific Session (APHRS2019)
  • Int'l Joint Research
• [Presentation] Algorithmic Auto-Recreation System of hiPSC-CMs Simulation and Prediction of Drug Testing. (2019)
  • Author(s): Hirohiko Kohjitani, Shigeya Kouda, Yukiko Himeno, Takeru Makiyama, Asami Kashiwa, Yimin Wuriyanghai, Yuta Yamamoto, Minoru Horie, Takeshi Kimura, Akinori Noma, Akira Amano
  • Organizer: 12th Asia Pacific Heart Rhythm Society Scientific Session (APHRS2019)
  • Int'l Joint Research
• [Presentation] Patients characteristics of idiopathic ventricular fibrillation carrying SCN5A mutations without Brugada or long QT syndrome. (2019)
  • Author(s): Takanori Aizawa, Takeru Makiyama, Hai Huang, Tomohiko Imamura, JingShan Gao, Yimin Wuriyanghai, Takeru Makiyama, Yuta Yamamoto, Hirohiko Kohjitani, Sayako Hirose, Jingshan Gao, Asami Kashiwa, Hai Huang, Wuriyanghai Yimin, Hirohiko Kohjitani, Yuta Yamamoto, Seiko Ohno, Minoru Horie
  • Organizer: 12th Asia Pacific Heart Rhythm Society Scientific Session (APHRS2019)
  • Int'l Joint Research
• [Presentation] Algorithmic Auto-Recreation System of hiPSC-CMs Simulation and Prediction of Drug Testing (2019)
  • Author(s): Hirohiko Kohjitani, Shigeya Kouda, Yukiko Himeno, Takeru Makiyama, Hai Huang, Jingshan Gao, Asami Kashiwa, Yimin Wuriyanghai, Daisuke Yoshinaga, Takeshi Kimura, Akira Amano.
  • Organizer: 第83回日本循環器学会学術集会
• [Presentation] Overlapping Sick Sinus Syndrome Associated with SCN5A Mutations in Patients with Brugada Syndrome (2019)
  • Author(s): Asami Kashiwa, Takeru Makiyama, Seiko Ohno, Hisaaki Aoki, Hirohiko Kohjitani Yimin Wuriyanghai, Yuta Yamamoto, Jingshan Gao, Huang Hai, Minoru Horie, Takeshi Kimura.
  • Organizer: 第83回日本循環器学会学術集会
• [Presentation] Three-Dimensional Structural Analysis of Mutant Ryanodine Receptor Type 2 Channels Associated with Catecholaminergic Polymorphic Ventricular Tachycardia. (2019)
  • Author(s): Jingshan Gao, Takeru Makiyama, Seiko Ohno, Yuta Yamamoto, Yimin Wuriyanghai, Hirohiko Kohjitani, Asami Kashiwa, Minoru Horie, Takeshi Kimura.
  • Organizer: 第83回日本循環器学会学術集会
• [Presentation] Modeling Overlapping Phenotypes of Long-QT and Brugada Syndrome with CACNA1C-E1115K that Alters Ion Selectivity of the Cardiac L-Type Calcium Channel Using Induced Pluripotent Stem Cells. (2019)
  • Author(s): Asami Kashiwa, Takeru Makiyama, Hirohiko Kohjitani, Yimin Wuriyanghai, Yuta Yamamoto, Jingshan Gao, Hai Huang, Taisuke Ishikawa, Seiko Ohno, Futoshi Toyoda, Seiichi Sato, Minoru Horie, Naomasa Makita, Takeshi Kimura
  • Organizer: 第66回日本不整脈心電学会学術大会
• [Presentation] Algorithmic Auto-Recreation System of hiPSC-CMs Simulation and Prediction of Drug Testing. (2019)
  • Author(s): Hirohiko Kohjitani, Shigeya Kouda, Yukiko Himeno, Takeru Makiyama, Hai Huang, Jingshan Gao, Asami Kashiwa, Yimin Wuriyanghai, Daisuke Yoshinaga, Takeshi Kimura, Akira Amano.
  • Organizer: 第66回日本不整脈心電学会学術大会
• [Presentation] Single Cell Electrophysiological Analysis of iPS Cell-derived Cardiomyocytes Generated from Long-QT Syndrome Patients Carrying a CALM2 Mutation Using a Membrane Potential Imaging System. (2019)
  • Author(s): Yuta Yamamoto, Takeru Makiyama(代発表), Yimin Wuriyanghai, Hirohiko Kohjitani, Sayako Hirose, Jingshan Gao, Asami Kashiwa, Huang Hai, Taisuke Ishikawa, Seiko Ohno, Kazuhisa Chonabayashi, Kenji Suda, Yoshinori Yoshida, Minoru Horie, Naomasa Makita, Takeshi Kimura,
  • Organizer: 第66回日本不整脈心電学会学術大会
• [Presentation] Structural Insights into Catecholaminergic Polymorphic Ventricular Tachycardia-associated RyR2 Mutant Channels using a Three-dimensional in Silico Model. (2019)
  • Author(s): Jingshan Gao, Takeru Makiyama, Seiko Ohno, Yuta Yamamoto, Yimin Wuriyanghai, Hirohiko Kohjitani, Asami Kashiwa, Hai Huang, Kouichi Katou, Minoru Horie, Takeshi Kimura.
  • Organizer: 第66回日本不整脈心電学会学術大会
• [Presentation] Phenotype-based High-throughput Classification of Long-QT Syndrome Subtypes Using Human Induced Pluripotent Stem Cells. (2019)
  • Author(s): Daisuke Yoshinaga, Shiro Baba, Takeru Makiyama, Hirofumi Shibata, Takuya Hirata, Kentaro Akagi, Koichi Matsuda, Hirohiko Kohjitani, Yimin Wuriyanghai, Katsutsugu Umeda, Yuta Yamamoto, Bruce R. Conklin, Minoru Horie, Junko Takita, Toshio Heike.
  • Organizer: 第66回日本不整脈心電学会学術大会
• [Presentation] Complex aberrant splicing in the induce pluripotent stem cell-derived cardiomyocytes from a patient with long-QT syndrome carrying KCNQ1-A344Aspl mutation. (2019)
  • Author(s): Wuriyanghai Y, Makiyama T, Sasaki K, Kamakura T, Yamamoto Y, Hayano M, Harita T, Nishiuchi S, Chen J, Kohjitani H, Hirose S, Yokoi F, Gao J, Chonabayashi K, Watanabe K, Ohno S, Yoshida Y, Kimura T, Horie M
  • Organizer: 第66回日本不整脈心電学会学術大会