Establishment of fundamental treatment for human genetic hearing loss

• Project/Area Number: 15H04989
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Otorhinolaryngology
• Research Institution: Kumamoto University
• Principal Investigator: Minoda Ryosei 熊本大学, 医学部附属病院, 非常勤診療医師 (30284772)
• Research Collaborator: MURAKAMI Daizo ; KUMAI Yoshihiko ; ISE Momoko
• Project Period (FY): 2015-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥17,160,000 (Direct Cost: ¥13,200,000、Indirect Cost: ¥3,960,000); Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2017: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000); Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2015: ¥11,830,000 (Direct Cost: ¥9,100,000、Indirect Cost: ¥2,730,000)
• Keywords: 遺伝子治療 / 胎生期治療 / Slc26a4遺伝子 / Tet systemベクター / Tet-on system / Slc26a4 / Cx30 / 遺伝性難聴 / 胎生期内耳 / エレクトロポレーション / 遺伝子難聴 / 内耳 / 胎仔 / マウス / 治療 / 耳胞 / 細胞移植 / ェレクトロポレーション

Outline of Final Research Achievements

Firstly, we constructed the plasmid vector which included Slc26a4 gene targeted short hairpin RNA with tet system. Then, that plasmid vector was injected and transferred into otocyst at embryonic day 11.5 according to our previous reported methods.
Doxycycline was administrated via ingestion between embryonic day 16 to postnatal day 6. As control, no Doxycycline group were used. Auditory assessment showed that hearing loss was detected in the treated mice, but not in the non-treated mice at postnatal day 30. Our results suggested that Slc26a4 gene expression between embryonic day 16 to postnatal day 6 was crucial for hearing. In the future, we investigate the Slc26a4 gene expression timing which is necessary for hearing.

Academic Significance and Societal Importance of the Research Achievements

遺伝子補充治療においては本来の遺伝子発現の時期と部位への厳密な一致は必要ないことが示唆される。このことを明らかにするために今回申請者は、これまで確立してきたマウス耳胞への遺伝子導入技術とテトラサイクリン系抗生剤の投与にて遺伝子発現の調整が可能なTet systemを組み合わせることにより、時期特異的な内耳遺伝子発現誘導マウスモデルを作製し研究を行った。本研究は、ヒト遺伝性難聴の主たる原因遺伝子であるSlc26a4をターゲットとして、機能欠失型変異による難聴に対する正常遺伝子補充治療の有効性とその限界を時間的・空間的視点から明らかにする一助となった。

Research Products

• [Journal Article] Engraftment of Human Pluripotent Stem Cell-derived Progenitors in the Inner Ear of Prenatal Mice (2018)
  • Author(s): Takeda Hiroki、Hosoya Makoto、Fujioka Masato、Saegusa Chika、Saeki Tsubasa、Miwa Toru、Okano Hideyuki、Minoda Ryosei
  • Journal Title: Scientific Reports Volume: 8 Issue: 1
  • DOI: 10.1038/s41598-018-20277-5
  • Peer Reviewed / Open Access
• [Journal Article] Protein transduction therapy into cochleae via the round window niche in guinea pigs. (2016)
  • Author(s): Takeda H, Kurioka T, Kaitsuka T, Tomizawa K, Matsunobu T,Hakim F, Mizutari K, Miwa T, Yamada T, Ise M, Shiotani A, Yumoto E, Minoda R
  • Journal Title: Mol Ther, Methods&Clin Dev. Volume: 3 Pages: 16055-16055
  • DOI: 10.1038/mtm.2016.55
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Presentation] Protein Transduction Therapy Via the Round Window in Guinea Pigs (2016)
  • Author(s): Minoda R
  • Organizer: ARO 39th Annual Midwinter Meeting
  • Place of Presentation: Sandiego
• [Presentation] Electroporation-Mediated Transuterine Gene Transfer into Mouse Otocysts (EUGO) utilizing NEPA21 electoroporator (2015)
  • Author(s): Minoda R
  • Organizer: 52th Inner Ear Biology Workshop
  • Place of Presentation: ローマ
• [Presentation] Roles of HGF/MET signaling in the mouse cochlea (2015)
  • Author(s): Miwa T
  • Organizer: 52th Inner Ear Biology Workshop
  • Place of Presentation: ローマ
• [Presentation] Mouse derived cell transplantation into the mouse otocyst in vivo (2015)
  • Author(s): Takeda H
  • Organizer: 52th Inner Ear Biology Workshop
  • Place of Presentation: ローマ
• [Remarks] 世界初 胎生期マウスの内耳への、ヒトiPS細胞由来細胞の移植に成功
  • URL: https://www.kumamoto-u.ac.jp/whatsnew/seimei/20180201