iPS細胞を用いた致死性遺伝性不整脈疾患の病態解明、治療法開発

2019 Fiscal Year Research-status Report

• Project/Area Number: 19K08538
• Research Institution: Kyoto University
• Principal Investigator: 牧山 武 京都大学, 医学研究科, 助教 (30528302)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: 不整脈 / 遺伝子 / iPS細胞 / 心筋細胞 / 心筋症 / 疾患モデル

Outline of Annual Research Achievements

当該年度は、遺伝性不整脈疾患の病態解明を目指し、以下の臨床、基礎研究を行った。
１．臨床研究：カテコラミン誘発性多形性心室頻拍（CPVT）は運動時、情動時の心室頻拍・細動、突然死を特徴とし、患者の約70%に心臓リアノジン受容体遺伝子（RYR2）変異が検出される。RyR2は巨大なタンパク質なため変異チャネルの機能解析が難しく、我々は、3D in silicoモデルを用いた病原性の検討を進めている。SWISS modelを用いて変異チャネルの3Dモデル作製を行い、立体構造の変化に関して検討を行ったところ、チャネル孔領域より遠位の変異においても構造変化によりチャネル孔内径の拡大を認め、筋小胞体からのカルシウムリークを引き起こしている可能性が示唆された。（欧州心臓病学会2019発表）本in silicoモデルは病原性が不明な変異"VUS"のアノテーションに寄与できると考え更なる検討を進めている。
２．基礎研究：遺伝性不整脈疾患のiPS細胞モデルを用いた解析。上記、RYR2遺伝子の変異が異なる表現型（CPVT、QT延長症候群）を呈する機序を解明するために、CRISPR-Cas9システムを用いたゲノム編集技術を用いて一塩基変異ノックインiPS細胞株を作製した。現在、Ca動態計測、活動電位計測により、疾患表現型を解析中である。また、L型カルシウムチャネルのイオン選択性に関わる部位に変異が検出されたQT延長症候群患者由来iPS細胞分化心筋細胞の解析において、イオン選択性低下による内向き電流の増加、再分極遅延、早期後脱分極（EAD）の所見を認め、ゲノム編集技術を用いた遺伝子変異修正株でこれらの機能異常がレスキューされることが分かった。本モデルを用いた治療薬の探索に関しては、薬効を認める既存薬を見出しており、引き続き検討を行っている。

Current Status of Research Progress

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

Reason

ゲノム編集技術を用いた一塩基置換iPS細胞株の作製、また、変異iPS細胞株のgene correctionに成功しており、進行状況はおおむね順調であると考える。

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、疾患発症機序の解明、治療法開発を目指した研究を進める。分化心筋細胞が静止膜電位が浅く自動能がある未熟心筋である点に関しては、パッチクランプ法を用いた活動電位記録時、人工的にIK1電流を加えるダイナミッククランプ法を用いて、より電気生理学的に成熟させた状態状態で検討を行っている。また、パッチクランプ法はスループットに難があり、膜電位色素(Fluovolt)を用いたハイスループットな薬効評価系を用いて効率よく機能解析を行う。

Causes of Carryover

少額の次年度使用額が生じたが、2020年度の研究（遺伝性不整脈iPS細胞モデルの解析）に使用する予定である。

Research Products

• [Journal Article] Optical Recording of Action Potentials in Human Induced Pluripotent Stem Cell-Derived Cardiac Single Cells and Monolayers Generated from Long QT Syndrome Type 1 Patients (2019)
  • Author(s): Takaki Tadashi、Inagaki Azusa、Chonabayashi Kazuhisa、Inoue Keiji、Miki Kenji、Ohno Seiko、Makiyama Takeru、Horie Minoru、Yoshida Yoshinori
  • Journal Title: Stem Cells International Volume: 2019 Pages: 1～12
  • DOI: 10.1155/2019/7532657
  • Peer Reviewed
• [Journal Article] Mutant KCNJ3 and KCNJ5 Potassium Channels as Novel Molecular Targets in Bradyarrhythmias and Atrial Fibrillation (2019)
  • Author(s): Yamada Noriaki、Asano Yoshihiro、Fujita Masashi、Yamazaki Satoru、Inanobe Atsushi、Matsuura Norio、Kobayashi Hatasu、Ohno Seiko、Ebana Yusuke、Tsukamoto Osamu、Ishino Saki、Takuwa Ayako、Kioka Hidetaka、Makiyama Takeru、Takashima Seiji、et al.
  • Journal Title: Circulation Volume: 139 Pages: 2157～2169
  • DOI: 10.1161/CIRCULATIONAHA.118.036761
  • Peer Reviewed
• [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] A de novo gain-of-function KCND3 mutation in early repolarization syndrome (2019)
  • Author(s): Takayama Koichiro、Ohno Seiko、Ding Wei-Guang、Ashihara Takashi、Fukumoto Daisuke、Wada Yuko、Makiyama Takeru、Kise Hiroaki、Hoshiai Minako、Matsuura Hiroshi、Horie Minoru
  • Journal Title: Heart Rhythm Volume: 16 Pages: 1698～1706
  • DOI: 10.1016/j.hrthm.2019.05.033
  • Peer Reviewed
• [Journal Article] ブルガダ症候群におけるアルコール代謝酵素の遺伝子変異 飲酒後の失神に関する知見(Genetic Variants of Alcohol-Metabolizing Enzymes in Brugada Syndrome: Insights into Syncope after Drinking Alcohol) (2019)
  • Author(s): Qi Wu,Hideki Hayashi,Daiki Hira,Keiko Sonoda,Satoshi Ueshima,Seiko Ohno,Takeru Makiyama,Tomohiro Terada,Toshiya Katsura,Katsuyuki Miura,Minoru Horie
  • Journal Title: Journal of Arrhythmia Volume: 35 Pages: 752-759
  • Peer Reviewed
• [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
• [Journal Article] Calmodulinopathy(カルモジュリン遺伝子関連不整脈疾患) iPS細胞技術を用いた疾患モデルの作製と遺伝子治療へのアプローチ (2019)
  • Author(s): 山本 雄大,牧山 武
  • Journal Title: 心電図 Volume: 39 Pages: 273-282
• [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
• [Presentation] Challenges of iPS cell-Based Disease Modeling and Therapeutic Approaches to Inherited Arrhythmias (2019)
  • Author(s): Takeru Makiyama
  • Organizer: 第 3回日本循環器学会基礎研究フォーラム
  • Invited
• [Patent(Industrial Property Rights)] アンチセンスオリゴヌクレオチド医薬のスクリーニング方法 (2020)
  • Inventor(s): 牧山武、山本雄大、入山友輔、近藤嘉紀、日高夕貴、石川龍太郎
  • Industrial Property Rights Holder: 牧山武、山本雄大、入山友輔、近藤嘉紀、日高夕貴、石川龍太郎
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 特願2020-036716
• [Patent(Industrial Property Rights)] ＣＡＬＭ２のアンチセンスオリゴヌクレオチド (2020)
  • Inventor(s): 入山友輔、近藤嘉紀、日高夕貴、石川龍太郎、牧山武、山本雄大
  • Industrial Property Rights Holder: 入山友輔、近藤嘉紀、日高夕貴、石川龍太郎、牧山武、山本雄大
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 特願2020-036715