難聴モデルマウス前庭の形態・機能評価と前庭を標的とした遺伝子導入の検討

2015 Fiscal Year Research-status Report

• Project/Area Number: 26462567
• Research Institution: Juntendo University
• Principal Investigator: 岡田 弘子 順天堂大学, 医学部, 助教 (20433774)
• Co-Investigator(Kenkyū-buntansha): 神谷 和作 順天堂大学, 医学部, 准教授 (10374159) ; 飯塚 崇 順天堂大学, 医学部, 助教 (40372932)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Keywords: 前庭機能 / Gjb2

Outline of Annual Research Achievements

コントロールマウス（C57BL/6）、Cx26-R75W変異を有するトランスジェニックマウス（Cx26R75W+）、コンディショナルノックアウトマウス(Cx26cKO)の3群において、幼若・成熟・老齢の前庭組織を摘出し、光学顕微鏡により有毛細胞不動毛の形態・支持細胞の形態学的解析を行った。現在のところは各群間での形態的な差異を認めなかった。また、コントロールマウス(C57BL/6)、R75W変位を有するCx26cKOの3群において、幼弱・成熟・老齢における前庭機能を評価した。現時点では各群での平衡機能検査間での有意差は認められていない。
Cx26cKOへのGJB2搭載アデノ随伴ウィルスへの投与を検討したところ、正円窓への投与、および半規管からの投与において前庭有毛細胞周囲の支持細胞への遺伝子導入が観察された。また、半規管からのウィルス液の還流法では大量のウィルス液を液循環により投与できるため、蝸牛頂部まで広範囲での遺伝子導入が可能であった。

Current Status of Research Progress

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

Reason

遺伝子改変モデル動物の分子病態解析（Kamiya,2014, J Clin Invest)、GJB2遺伝子治療(Iizuka, 2015, Human Moleculare Genetics)いずれにおいても有意義な成果を挙げることができたため。

Strategy for Future Research Activity

遺伝子改変モデル動物の分子病態解析（Kamiya,2014, J Clin Invest)、GJB2遺伝子治療(Iizuka, 2015, Human Moleculare Genetics)いずれにおいても有意義な成果を挙げることができたが、今後はこれらの成果をもとにして前庭の病態メカニズムを解明し、遺伝子治療によって予防、改善が可能な有効な治療法を確立することを目指す。

Causes of Carryover

予定していた試薬の購入を備蓄品の使用で代用し、予定金額を使用しなかったため。

Expenditure Plan for Carryover Budget

今後は毎月、会計担当者と共に残額の確認と使用資金の検討を行うことにより効率的に試薬消耗品の購入を行い、研究課題が最大限に円滑に進むための運用計画を遂行する。

Research Products

• [Journal Article] Postnatal Development and Maturation of the Vestibular Organ in Dominant-Negative Connexin 26 Transgenic Mouse. (2015)
  • Author(s): Hiroko Okada, Kazusaku Kamiya., Takashi Iizuka and Katsuhisa Ikeda.
  • Journal Title: J Otol Rhinol Volume: S1 Pages: 37-40
  • DOI: 10.4172/2324-8785.S1-009
  • Peer Reviewed / Open Access
• [Journal Article] Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26 Deficient Mice. (2015)
  • Author(s): Anzai, T., Fukunaga, I., Hatakeyama, K., Fujimoto, A., Kobayashi, K., Nishikawa, A., Aoki, T., Noda, T., Minowa, O., Ikeda, K., and Kamiya, K
  • Journal Title: PloS one Volume: 10 Pages: e0141258
  • DOI: 10.1371/journal.pone.0141258.
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Differentiation of iPS Cells to Cochlear Cells are Regulated Depending on the Part of Co-cultured Organs. (2015)
  • Author(s): Kazusaku Kamiya, Keiko Karasawa, Kazuma Kobayashi, Asuka Miwa and Katsuhisa Ikeda.
  • Journal Title: J Otol Rhinol Volume: S1 Pages: 34-36
  • DOI: 10.4172/2324-8785.S1-008
  • Peer Reviewed / Open Access
• [Journal Article] Perinatal Gjb2 gene transfer rescues hearing in a mouse model of hereditary deafness. (2015)
  • Author(s): Iizuka, T., Kamiya, K., Gotoh, S., Sugitani, Y., Suzuki, M., Noda, T., Minowa, O., and Ikeda, K.
  • Journal Title: Human molecular genetics Volume: 24 Pages: 3651-3661
  • DOI: 10.1093/hmg/ddv109.
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Connexin26 regulates assembly and maintenance of cochlear gap junction macromolecular complex for normal hearing (2015)
  • Author(s): Kazusaku Kamiya, Ichiro Fukunaga, Kaori Hatakeyama and Katsuhisa Ikeda
  • Journal Title: AIP Conference Proceedings Volume: 1703 Pages: 30018;1-3
  • DOI: 10.1063/1.4939333
  • Acknowledgement Compliant
• [Presentation] GJB2変異遺伝性難聴に対する細胞治療・遺伝子治療法の開発 (2016)
  • Author(s): 神谷 和作
  • Organizer: 第15回日本再生医療学会総会
  • Place of Presentation: 大阪国際会議場(大阪市)
  • Year and Date: 2016-03-17 – 2016-03-19
• [Presentation] iPS細胞からのConnexin26発現細胞への分化誘導と細胞間ギャップ結合プラークの構築 (2016)
  • Author(s): 福永一朗、畠山佳欧里、 青木徹、藤本あゆみ、 西川貴菜、美野輪治、 池田 勝久、神谷和作
  • Organizer: 第15回日本再生医療学会総会
  • Place of Presentation: 大阪国際会議場(大阪市)
  • Year and Date: 2016-03-17 – 2016-03-19
• [Presentation] コネキシン26欠損マウスからのiPS細胞の樹立 (2016)
  • Author(s): 青木徹、福永一朗、 畠山佳欧里、藤本あゆみ、西川貴菜、美野輪治、 池田勝久、神谷和作
  • Organizer: 第15回日本再生医療学会総会
  • Place of Presentation: 大阪国際会議場(大阪市)
  • Year and Date: 2016-03-17 – 2016-03-19
• [Presentation] 内耳フィーダー細胞を用いたiPS細胞の分化誘導によるCx26ギャップ結合形成細胞の樹立 (2016)
  • Author(s): 藤本あゆみ、福永一朗、 畠山佳欧里、青木徹、 西川貴菜、池田勝久、 神谷 和作
  • Organizer: 第15回日本再生医療学会総会
  • Place of Presentation: 大阪国際会議場(大阪市)
  • Year and Date: 2016-03-17 – 2016-03-19
• [Presentation] Restoration og Cochlear Gap Junction for GJB2 (2016)
  • Author(s): Kazusaku Kamiya, Ichiro Fukunaｇa
  • Organizer: Association for Research in Otolaryngology (ARO), 39th Annual MidWinter Meeting
  • Place of Presentation: Manchester Grand Hyatt, San Diego, CA, USA
  • Year and Date: 2016-02-20 – 2016-02-24
  • Int'l Joint Research
• [Presentation] Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26-Deficient Mice (2016)
  • Author(s): Takashi Anzai, Kazusaku Kamiya, Katsuhisa Ikeda
  • Organizer: Association for Research in Otolaryngology (ARO), 39th Annual MidWinter Meeting
  • Place of Presentation: Manchester Grand Hyatt, San Diego, CA, USA
  • Year and Date: 2016-02-20 – 2016-02-24
  • Int'l Joint Research
• [Presentation] Generation of induced pluripoten stem cells form connexin26 conditional knock out mouce (2016)
  • Author(s): Toru Aoki, Katsuhisa Ikeda, Kazusaku Kamiya
  • Organizer: Association for Research in Otolaryngology (ARO), 39th Annual MidWinter Meeting
  • Place of Presentation: Manchester Grand Hyatt, San Diego, CA, USA
  • Year and Date: 2016-02-20 – 2016-02-24
  • Int'l Joint Research
• [Presentation] Differentiation of mouse iPS cell into Cx26-positive cell and formation of inter cellular Cx26-gap junction plaque (2016)
  • Author(s): Ichiro Fukunaga, Kaori Hatakeyama,Toru Aoki, Atena Nishikawa, Ayumi Fujimoto, Katsuhisa Ikeda, Kazusaku Kamiya
  • Organizer: Association for Research in Otolaryngology (ARO), 39th Annual MidWinter Meeting
  • Place of Presentation: Manchester Grand Hyatt, San Diego, CA, USA
  • Year and Date: 2016-02-20 – 2016-02-24
  • Int'l Joint Research
• [Presentation] Induction of iPS cell differentiation into Connexin26 positive cells with gap junction plaques using cochlear feeder cell (2016)
  • Author(s): Ayumi Fujimoto, Ichiro Fukunaga, Kaori Hatakeyama, Toru Aoki, Atena Nishikawa, Katsuhisa Ikeda, Kazusaku Kamiya
  • Organizer: Association for Research in Otolaryngology (ARO), 39th Annual MidWinter Meeting
  • Place of Presentation: Manchester Grand Hyatt, San Diego, CA, USA
  • Year and Date: 2016-02-20 – 2016-02-24
  • Int'l Joint Research
• [Presentation] Various cochlear gap junction plaque reproduced in human cell line (2016)
  • Author(s): Kaori Hatakeyama, Katsuhisa Ikeda, Kazusaku Kamiya
  • Organizer: Association for Research in Otolaryngology (ARO), 39th Annual MidWinter Meeting
  • Place of Presentation: Manchester Grand Hyatt, San Diego, CA, USA
  • Year and Date: 2016-02-20 – 2016-02-24
  • Int'l Joint Research
• [Presentation] Cochlear gap junction plaque, stabilized macromolecular complex composed of specific connexins (2015)
  • Author(s): Kazusaku Kamiya, Ichiro Fukunaga, Kaori Hatakeyama, Toru Aoki, Ayumi Fujimoto, Atena Nishikawa ,Takashi Anzai, Osamu Minowa, Katsuhisa Ikeda
  • Organizer: 52nd Inner Ear Biology Workshop
  • Place of Presentation: Campus of the Catholic University of Rome, Italy
  • Year and Date: 2015-09-12 – 2015-09-15
  • Int'l Joint Research
• [Presentation] Caveolins accumulates at the organ of Corti in GJB2 associated deafness (2015)
  • Author(s): Takashi Anzai, Kazusaku Kamiya, Katsuhisa Ikeda
  • Organizer: 52nd Inner Ear Biology Workshop
  • Place of Presentation: Campus of the Catholic University of Rome, Italy
  • Year and Date: 2015-09-12 – 2015-09-15
  • Int'l Joint Research
• [Presentation] Reproduction of various types of cochlear gap junction plaques in human cell line (2015)
  • Author(s): Kaori Hatakeyama, Katsuhisa Ikeda, Kazusaku Kamiya
  • Organizer: 52nd Inner Ear Biology Workshop
  • Place of Presentation: Campus of the Catholic University of Rome, Italy
  • Year and Date: 2015-09-12 – 2015-09-15
  • Int'l Joint Research
• [Presentation] Differentiation of mouse iPS cell into Cx26-positive cell and formation of inter cellular Cx26-gap junction plaque (2015)
  • Author(s): Ichiro Fukunaga, Kaori Hatakeyama,Toru Aoki, Atena Nishikawa, Ayumi Fujimoto, Osamu Minowa, Katsuhisa Ikeda, Kazusaku Kamiya
  • Organizer: 52nd Inner Ear Biology Workshop
  • Place of Presentation: Campus of the Catholic University of Rome, Italy
  • Year and Date: 2015-09-12 – 2015-09-15
  • Int'l Joint Research