Development of Human Disease Model of Arrhythmogenic Right Ventricular Cardiomyopathy for Personalized Medicine

• Project/Area Number: 20K21602
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 53:Organ-based internal medicine and related fields
• Research Institution: Osaka University
• Principal Investigator: Sakata Yasushi 大阪大学, 医学系研究科, 教授 (00397671)
• Co-Investigator(Kenkyū-buntansha): 肥後 修一朗 大阪大学, 医学系研究科, 特任准教授(常勤) (00604034) ; 彦惣 俊吾 大阪大学, 医学系研究科, 准教授 (30423164)
• Project Period (FY): 2020-07-30 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2021: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000); Fiscal Year 2020: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
• Keywords: 不整脈原性心筋症 / 疾患iPS細胞 / ゲノム編集 / iPS細胞 / 遺伝子治療 / 不整脈源性右室心筋症

Outline of Research at the Start

不整脈源性右室心筋症(ARVC)は、重篤な不整脈を来す難病です。現在のところ、この病気の進行を予防できる有効な治療法が存在しません。細胞と細胞をつなぐ介在板を構成するタンパク質のひとつであるプラコフィリン2(PKP2)はARVCの主要な原因遺伝子の一つです。本研究開発では、PKP2をターゲット遺伝子とし、疾患iPS細胞技術とゲノム編集技術を組み合わせ、ARVCを再現するヒトモデル細胞の樹立と、モデル細胞を用いた病態の解明、治療法の開発を目指します。介在板異常を対象に、その疾患メカニズムの解明を試み、より幅広い心筋症の分類、個々の病気に応じた個別化医療につなげることを目指します。

Outline of Final Research Achievements

Loss-of-function mutations in PKP2, encoding plakophilin-2, cause arrhythmogenic cardiomyopathy (AC). Here, we generated isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with precisely adjusted expression of plakophilin-2 from a patient with AC carrying a heterozygous frameshift PKP2 mutation. After monolayer differentiation, plakophilin-2 deficiency led to reduced contractility, disrupted intercalated disc structures, and impaired desmosome assembly in iPSC-CMs. Allele-specific fluorescent labeling of endogenous DSG2 in the generated isogenic lines enabled real-time desmosome-imaging under an adjusted dose of plakophilin-2. AAV-mediated gene replacement of PKP2 recovered contractility and restored desmosome assembly, which was sequentially captured by desmosome-imaging in plakophilin-2-deficient iPSC-CMs. Our isogenic set of iPSC-CMs recapitulates AC pathology and provides a rapid and convenient cellular platform for therapeutic development.

Academic Significance and Societal Importance of the Research Achievements

不整脈原性心筋症には既存の心不全治療薬が奏功せず、進行した場合、心臓移植しか治療方法が残されていません。本研究ではプラコフィリン2タンパク質が欠損したiPS細胞由来分化心筋細胞を用いて、その病態解明を行い、遺伝子補充の治療概念を実証しました。精密に遺伝子を改変した不整脈原性心筋症モデル細胞、及びデスモゾームを可視化するイメージング細胞は、難治性心筋症に対する今後の治療法開発において、有用なプラットフォームを提供すると考えられます。

Research Products

• [Journal Article] Phospholamban p.Arg14del Cardiomyopathy: A Japanese Case Series (2022)
  • Author(s): Tabata Tomoka、Kuramoto Yuki、Ohtani Tomohito、Miyawaki Hiroshi、Miyashita Yohei、Sera Fusako、Kioka Hidetaka、Higo Shuichiro、Asano Yoshihiro、Hikoso Shungo、Sakata Yasushi
  • Journal Title: Internal Medicine Volume: 61 Issue: 13 Pages: 1987-1993
  • DOI: 10.2169/internalmedicine.8594-21
  • NAID: 130008130223
  • ISSN: 0918-2918, 1349-7235
  • Year and Date: 2022-07-01
  • Peer Reviewed / Open Access
• [Journal Article] Modeling reduced contractility and impaired desmosome assembly due to plakophilin-2 deficiency using isogenic iPS cell-derived cardiomyocytes (2022)
  • Author(s): Inoue Hiroyuki、Nakamura Satoki、Higo Shuichiro、Shiba Mikio、Kohama Yasuaki、Kondo Takumi、Kameda Satoshi、Tabata Tomoka、Okuno Shota、Ikeda Yoshihiko、Li Junjun、Liu Li、Yamazaki Satoru、Takeda Maki、Ito Emiko、Takashima Seiji、Miyagawa Shigeru、Sawa Yoshiki、Hikoso Shungo、Sakata Yasushi
  • Journal Title: Stem Cell Reports Volume: 17 Issue: 2 Pages: 337-351
  • DOI: 10.1016/j.stemcr.2021.12.016
  • Peer Reviewed / Open Access
• [Journal Article] Genome Editing in Human Induced Pluripotent Stem Cells (hiPSCs) (2021)
  • Author(s): Higo Shuichiro、Hikoso Shungo、Miyagawa Shigeru、Sakata Yasushi
  • Journal Title: Methods Mol Biol Volume: - Pages: 235-245
  • DOI: 10.1007/978-1-0716-1484-6_21
  • ISBN: 9781071614839, 9781071614846
• [Journal Article] Phenotypic recapitulation and correction of desmoglein-2-deficient cardiomyopathy using human-induced pluripotent stem cell-derived cardiomyocytes (2021)
  • Author(s): Shiba Mikio、Higo Shuichiro、Kondo Takumi、Li Junjun、Liu Li、Ikeda Yoshihiko、Kohama Yasuaki、Kameda Satoshi、Tabata Tomoka、Inoue Hiroyuki、Nakamura Satoki、Takeda Maki、Ito Emiko、Takashima Seiji、Miyagawa Shigeru、Sawa Yoshiki、Hikoso Shungo、Sakata Yasushi
  • Journal Title: Human Molecular Genetics Volume: 30 Issue: 15 Pages: 1384-1397
  • DOI: 10.1093/hmg/ddab127
  • Peer Reviewed / Open Access
• [Journal Article] Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes (2020)
  • Author(s): Kohama Yasuaki、Higo Shuichiro、Masumura Yuki、Shiba Mikio、Kondo Takumi、Ishizu Takamaru、Higo Tomoaki、Nakamura Satoki、Kameda Satoshi、Tabata Tomoka、Inoue Hiroyuki、Motooka Daisuke、Okuzaki Daisuke、Takashima Seiji、Miyagawa Shigeru、Sawa Yoshiki、Hikoso Shungo、Sakata Yasushi
  • Journal Title: Scientific Reports Volume: 10 Issue: 1 Pages: 15348-15348
  • DOI: 10.1038/s41598-020-72216-y
  • Peer Reviewed / Open Access
• [Presentation] Modeling Reduced Contractility and Impaired Desmosome Assembly due to Plakophilin-2 Deficiency using Isogenic iPS Cell-Derived Cardiomyocytes (2022)
  • Author(s): Hiroyuki Inoue,Satoki Nakamura,Shuichiro Higo,Mikio Shiba,Yasuaki Kohama,Takumi Kondo,Satoshi Kameda,Tomoka Tabata,Shota Okuno,Yoshihiko Ikeda,Junjun Li,Li Liu,Satoru Yamazaki,Maki Takeda,Emiko Ito,Seiji Takashima,Shigeru Miyagwa,Sawa Yoshiki,Shungo Hikoso,Yasushi Sakata
  • Organizer: 第86回 日本循環器学会学術集会 YIA (Basic Research部門）優秀賞
• [Presentation] Development of Personalized Medicine Targeting Arrhythmogenic Cardiomyopathy Concealed in Advanced Heart Failure (2022)
  • Author(s): Shuichiro Higo
  • Organizer: 第 86 回日本循環器学会学術集会 幹細胞・遺伝子治療・心臓再生の最前線 Cutting-edge Science in Stem Cell, Gene Therapy, and Regeneration for Cardiovascular Disease
• [Presentation] 悪性心室性頻拍と重症心不全を特徴とする 日本におけるホスホランバン p.Arg14del心筋症の臨床像 (2021)
  • Author(s): 田端 智香,藏本 勇希,大谷 朋仁,宮脇 大,宮下 洋平,世良 英子,木岡 秀隆,肥後 修一朗,朝野 仁裕,彦惣 俊吾,坂田 泰史
  • Organizer: 第25回日本心不全学会学術集会
• [Presentation] Modeling Augmented Sarcomeric Calcium Retention and Altered Calcium Handling Pathway by TNNT2 Δ160E Mutation using Isogenic Human iPS Cell-derived Cardiomyocytes (2021)
  • Author(s): Takumi Kondo,Shuichiro Higo,Mikio Shiba,Yasuaki Kohama,Satoshi Kameda,Tomoka Tabata,Hiroyuki Inoue,Shota Okuno,Maki Takeda,Yuki Kuramoto,Jong-Kook Lee,Seiji Takashima,Shigeru Miyagawa,Shungo Hikoso,Yasushi Sakata
  • Organizer: 第25回日本心不全学会学術集会YIA基礎部門優秀賞
• [Presentation] Strategies to Promote Homology-directed Repair in Post-mitotic Cardiomyocytes (2021)
  • Author(s): Shuichiro Higo,Yasushi Sakata
  • Organizer: ESC CONGRESS 2021 symposium CRISPR/Cas9 gene editing for heart disease: ready for prime time?
  • Int'l Joint Research / Invited
• [Presentation] 難治性心筋症に対する個別化医療 (2021)
  • Author(s): 肥後修一朗
  • Organizer: 第7回日本心筋症研究会 シンポジウム4「心筋症のゲノム研究最前線」
  • Invited
• [Presentation] トロポニンTΔ160E変異を有するアイソジェニックiPS心筋を用いた拡張相肥大型心筋症の病態解明 (2021)
  • Author(s): 近藤匠巳,肥後修一朗,志波幹夫,小濱康明,亀田聡士,田端智香,井上裕之,奥野翔太,武田真季,藏本勇希,李鍾國,高島成二,宮川繁,彦惣俊吾,坂田泰史
  • Organizer: 第7回日本心筋症研究会
• [Presentation] Development of Precision Medicine for Arrhythmogenic Cardiomyopathy using Isogenic Human Disease Model (2021)
  • Author(s): Shuichiro Higo,Shungo Hikoso,Yasushi Sakata
  • Organizer: 第85日本循環器学会学術集会
• [Presentation] Drug discovery for cardiomyopathies using disease-specific iPSCs (2020)
  • Author(s): Shuichiro Higo,Shungo Hikoso,Shigeru Miyagawa,Yoshiki Sawa,Yasushi Sakata
  • Organizer: 第84回日本循環器学会学術集会 Modelling Cardiovascular Disease with Human iPS Cells: Current Status and Future Prospects
  • Invited
• [Presentation] Generation of Human Disease Model for Personalized Medicine Targeting Intractable Cardiomyopathy (2020)
  • Author(s): Shuichiro Higo, Shungo Hikoso, Shigeru Miyagawa, Yoshiki Sawa, Yasushi Sakata
  • Organizer: 第84回日本循環器学会学術集会 ESC-JCS Young Researchers Session
  • Invited
• [Presentation] 不整脈源性右室心筋症（ARVC）を迅速に再現するヒトモデル細胞の樹立 (2020)
  • Author(s): 中村 聡希,肥後 修一朗,志波 幹夫,小濱 康明,近藤 匠巳,亀田 聡士,田端 智香,井上 裕之,山崎 悟,武田 真季,高島 成二,宮川 繁,澤 芳樹,彦惣 俊吾,坂田 泰史
  • Organizer: 第6回日本心筋症研究会
• [Presentation] Phenotypic Recapitulation and Correction of Desmoglein-2-deficient Cardiomyopathy using Human Induced Pluripotent Stem Cellderived Cardiomyocytes (2020)
  • Author(s): 志波幹夫,肥後修一朗,近藤匠巳,李俊君,劉莉,小濱康明,亀田聡士,田端智香,井上裕之,中村聡希,武田真季,池田善彦,高島成二,宮川繁,澤芳樹,彦惣俊吾,坂田泰史
  • Organizer: 第68回日本心臓病学会
• [Presentation] ハイコンテントイメージング・数値計算ソフトウェアを用いた心筋症iPS 分化心筋におけるカルシウム動態解析 (2020)
  • Author(s): 田端智香,肥後修一朗,増村雄喜,志波幹夫,小濱康明,近藤匠巳,亀田聡士,井上裕之,中村聡希,高島成二,宮川繁,澤芳樹,彦惣俊吾,坂田泰史
  • Organizer: 第6回日本心筋症研究会
• [Presentation] Adeno-associated Virus-mediated Gene Delivery Promotes S-Phase Entry-independent Homology-directed Repair in Cardiomyocytes (2020)
  • Author(s): Yasuaki Kohama,Shuichiro Higo,Mikio Shiba,Takumi Kondo,Satoki Nakamura,Satoshi Kameda,Tomoka Tabata,Hiroyuki Inoue,Seiji Takashima,Shigeru Miyagawa,Yoshiki Sawa,Shungo Hikoso,Yasushi Sakata
  • Organizer: 第24回日本心不全学会学術集会
• [Book] Heart View 疾患iPS細胞を用いた心筋症個別化医療へのアプローチ (2022)
  • Author(s): 肥後 修一朗
  • Publisher: MEDICAL VIEW
• [Book] 医学のあゆみ (2021)
  • Author(s): 肥後修一朗
  • Total Pages: 2
  • Publisher: 医歯薬出版
• [Remarks] 重症心不全内科治療学グループ
  • URL: http://www.cardiology.med.osaka-u.ac.jp/?page_id=33635