Basic studies toward CRISPR/Cas9-mediated cell therapy

• Project/Area Number: 26461530
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Pediatrics
• Research Institution: Kitasato University
• Principal Investigator: Miyashita Toshiyuki 北里大学, 医学部, 教授 (60174182)
• Co-Investigator(Renkei-kenkyūsha): NAGAO Kazuaki 北里大学, 医学部, 講師 (60392487) ; KAMEYAMA Kohzoh 北里大学, 医学部, 講師 (40214556) ; TAKAYAMA Yoshinaga 北里大学, 医学部, 講師 (90245407)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2015: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2014: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: 母斑基底細胞癌症候群 / CRISPR/Cas9システム / iPS細胞 / 遺伝子編集 / PTCH1 / 髄芽腫 / LOH / モザイク / CRISPR/Cas9

Outline of Final Research Achievements

We have successfully established nevoid basal cell carcinoma syndrome (NBCCS)-specific induced pluripotent stem cells (iPS cells). Medulloblastoma was formed in all the teratomas generated from all NBCCS-specific iPSC clones established from 4 different patients. These results demonstrated that NBCCS-specific iPSCs can be a good model of NBCCS-derived medulloblastoma, and thereby used for drug screening to prevent or treat medulloblastomas.
In addition, by using CRISPR/Cas9 system, we also generated iPS cells in which mutation was introduced in remaining normal PTCH1 allele. These cells exhibited accelerated cell growth and expression of Gli target genes indicating up-regulation of hedgehog signaling. Moreover, we generated iPS cells in which mutated allele was edited and corrected.

Research Products

• [Journal Article] Somatic mosaicism containing double mutations in PTCH1 revealed by generation of induced pluripotent stem cells from nevoid basal cell carcinoma syndrome. (2017)
  • Author(s): Ikemoto, Y., Takayama, Y., Fujii, K., Masuda, M., Kato, C., Hatsuse, H., Fujitani, K., Nagao, K., Kameyama, K., Ikehara, H., Toyoda, M., Umezawa, A. and Miyashita, T.
  • Journal Title: Journal of Medical Genetics Volume: － Issue: 8 Pages: 579-584
  • DOI: 10.1136/jmedgenet-2016-104490
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Brain morphology in children with nevoid basal cell carcinoma syndrome. (2017)
  • Author(s): Shiohama, T., Fuji, K., Miyashita, T., Mizuochi, H., Uchikawa, H., Shimojo, N.
  • Journal Title: American Journal of Medical Genetics A Volume: 173 Issue: 4 Pages: 946-952
  • DOI: 10.1002/ajmg.a.38115
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Nevoid basal cell carcinoma syndrome caused by splicing mutations in the PTCH1 gene. (2017)
  • Author(s): Kato, C., Fujii, K., Arai, Y., Hatsuse, H., Nagao, K., Takayama, Y., Kameyama, K., Fujii, K., and Miyashita, T.
  • Journal Title: Familial Cancer Volume: 16 Issue: 1 Pages: 131-138
  • DOI: 10.1007/s10689-016-9924-2
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Gorlin syndrome with an ovarian leiomyoma associated with a PTCH1 second hit (2016)
  • Author(s): Akizawa, Y., Miyashita, T., Sasaki, R., Nagata, R., Aoki, R., Ishitani, K., Nagashima, Y., Matsui, H., Saito, K
  • Journal Title: Am J Med A Volume: 170 Issue: 4 Pages: 1029-1034
  • DOI: 10.1002/ajmg.a.37517
  • Peer Reviewed / Open Access
• [Journal Article] Chromatin condensation of Xist genomic loci during oogenesis in mice (2015)
  • Author(s): Fukuda, A., Mitani, A., Miyashita, T., Umezawa, A., Akutsu, H
  • Journal Title: Development Volume: 142 Pages: 4049-4055
  • DOI: 10.1242/dev.127308
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A rare case of a symptomatic tumor found in the groin area: An atypical location unexposed to the known causes (2015)
  • Author(s): Toyonaga, E., Hata, H., Nakayama, C., Homma, E., Miyashita, T., Shimizu, H
  • Journal Title: Case Rep. Oncol Volume: 8 Issue: 3 Pages: 536-539
  • DOI: 10.1159/000442148
  • NAID: 120006363266
  • Peer Reviewed / Open Access
• [Journal Article] Transient abnormal myelopoiesis of extremely immature infant followed by blast cell resurgence two months later: a case study. (2015)
  • Author(s): Nomura, Y., Miyauchi, J., Ota, E., Yanai, F., Miyashita, T., Terui, K., Ito, E. and Hirose, S.
  • Journal Title: Jpn. J. Pediatr. Hematol. Oncol. Volume: 52 Pages: 36-39
• [Journal Article] PTCH1, PTCH2, SUFU (2015)
  • Author(s): 宮下俊之
  • Journal Title: 日本臨牀 Volume: 73 Pages: 375-379
• [Journal Article] Mirror syndrome associated with fetal transient abnormal myelopoiesis in Down syndrome (2015)
  • Author(s): Kobayashi, Y., Miyoshi, T., Matsuyama, T., Miyauchi, J., Miyashita, T., Ishibashi-Ueda, H. and Yoshimatsu, J.
  • Journal Title: Pathology International Volume: 印刷中 Issue: 8 Pages: 443-445
  • DOI: 10.1111/pin.12291
  • Peer Reviewed / Open Access
• [Journal Article] Gorlin syndrome (nevoid basal cell carcinoma syndrome): update and literature review (2014)
  • Author(s): Fujii K, Miyashita T
  • Journal Title: Pediatric International Volume: 56 Issue: 5 Pages: 667-674
  • DOI: 10.1111/ped.12461
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] 角化嚢胞性歯原性腫瘍を合併したB前駆細胞型急性リンパ性白血病の一例 (2016)
  • Author(s): 渡邊敦、杣津晋平、大城浩子、赤羽弘資、合井久美子、犬飼岳史、藤井克則、宮下俊之、杉田完爾
  • Organizer: 第58回日本小児血液・がん学会学術集会
  • Place of Presentation: 品川プリンスホテル（東京都港区）
  • Year and Date: 2016-12-15
• [Presentation] Gorlin症候群患者由来iPS細胞を用いた髄芽腫の作製 (2016)
  • Author(s): 池本 優、豊田 雅士、那須 道世、初瀬 洋美、長尾 和右、高山 吉永、亀山 孝三、梅澤 明弘、宮下 俊之
  • Organizer: 第39回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（横浜市）
  • Year and Date: 2016-11-30
• [Presentation] Entire PTCH1 deletions detected in 9 families with nevoid basal cell carcinoma syndrome (2016)
  • Author(s): 高山吉永、長尾一右、藤井克則、宮下俊之
  • Organizer: 第75回日本癌学会学術総会
  • Place of Presentation: パシフィコ横浜（横浜市）
  • Year and Date: 2016-10-06
• [Presentation] Gorlin症候群における脳形態解析－ヘッジホッグシグナルのヒト脳への影響－ (2016)
  • Author(s): 塩浜直、藤井克則、宮下俊之、内川英紀、水落弘美、池原甫、福原知之、下条直樹
  • Organizer: 第58回日本小児神経学会学術集会
  • Place of Presentation: 京王プラザホテル（東京都新宿区）
  • Year and Date: 2016-06-03
• [Presentation] 母斑基底細胞癌症候群におけるスプライシング変異の検討 (2016)
  • Author(s): 宮下俊之、藤井克則
  • Organizer: 第22回日本家族性腫瘍学会学術集会
  • Place of Presentation: ひめぎんホール(愛媛県松山市)
  • Year and Date: 2016-06-03
• [Presentation] CRISPR/Cas9システムを用いた2点同時切断による目的ゲノム領域除去細胞株の作製 (2015)
  • Author(s): 加藤千勢、長尾和右、初瀬洋美、高山吉永、亀山孝三、宮下俊之
  • Organizer: 第38回日本分子生物学会年会
  • Place of Presentation: 神戸国際会議場（兵庫県神戸市）
  • Year and Date: 2015-12-01
• [Presentation] Establishment of the cell lines with a large genomic deletion by CRISPR/Cas9 system. (2015)
  • Author(s): 長尾和右、高山吉永、宮下俊之
  • Organizer: 第74回日本癌学会学術総会
  • Place of Presentation: 名古屋国際会議場（愛知県名古屋市）
  • Year and Date: 2015-10-08
• [Presentation] CRISPR/Cas9システムを用いたSNPジェノタイプ転換の試み (2014)
  • Author(s): 高山吉永 初瀬洋美 中島早耶 鈴木香織 加藤千勢 長尾和右 亀山孝三 宮下俊之
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2014-11-25 – 2014-11-27
• [Presentation] CRISPR/Cas9システムを用いた広域欠損ゲノムを有する細胞株の作製 (2014)
  • Author(s): 長尾和右 加藤千勢 初瀬洋美 高山吉永 亀山孝三 宮下俊之
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2014-11-25 – 2014-11-27
• [Presentation] 卵巣腫瘍においてPTCH1遺伝子の セカンドヒットを同定したGorlin症候群の1例 (2014)
  • Author(s): 秋澤叔香 宮下俊之 佐々木亮 浦野真理 佐藤裕子 青木亮子 石谷健 山内あけみ 松井英雄 齋藤加代子
  • Organizer: 日本人類遺伝学会第59回大会
  • Place of Presentation: タワーホール船堀（東京都江戸川区）
  • Year and Date: 2014-11-19 – 2014-11-22
• [Presentation] CRISPR/Cas9システムを用いたソニックヘッジホッグシグナル伝達系構成遺伝子ノックアウト細胞株の作製 (2014)
  • Author(s): 長尾和右 高山吉永 宮下俊之
  • Organizer: 第73回日本癌学会学術総会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] CRISPR/Cas9システムによる欠失導入効率の検討 (2014)
  • Author(s): 加藤千勢 高山吉永 亀山孝三 初瀬洋美 長尾和右 宮下俊之
  • Organizer: 第27回北里大学バイオサイエンスフォーラム
  • Place of Presentation: 北里大学（神奈川県相模原市）
  • Year and Date: 2014-08-21 – 2014-08-22
• [Presentation] Hedgehog signaling is synergistically enhanced by nutritional deprivation and ligands stimulation in human fibroblasts of Gorlin syndrome (2014)
  • Author(s): Mizuochi, H., Fujii, K., Shiohama, T., Uchikawa, H., Miyashita, T. and Shimojo, N
  • Organizer: Hedgehog 2014
  • Place of Presentation: Ann Arbor, USA
  • Year and Date: 2014-08-04 – 2014-08-08
• [Presentation] Oxidative stress-induced JNK1 phosphorylation inhibits hedgehog signaling and osteoblast differentiation (2014)
  • Author(s): Fujii, K., Shiohama, T., Mizuochi, H., Uchikawa, H., Takatani, T., Kohno, Y. and Miyashita, T
  • Organizer: Hedgehog 2014
  • Place of Presentation: Ann Arbor, USA
  • Year and Date: 2014-08-04 – 2014-08-08
• [Presentation] 母斑基底細胞癌症候群に発症した腫瘍の遺伝子解析 (2014)
  • Author(s): 宮下俊之 藤井克則
  • Organizer: 第20回日本家族性腫瘍学会学術集会
  • Place of Presentation: コラッセふくしま（福島県福島市）
  • Year and Date: 2014-06-13 – 2014-06-14
• [Book] 最新遺伝性腫瘍・家族性腫瘍研究と遺伝カウンセリング (2016)
  • Author(s): 宮下俊之、藤井克則
  • Total Pages: 336
  • Publisher: メディカルドゥ
• [Book] Methods in Molecular Biology 1278 (2015)
  • Author(s): Miyashita, T.
  • Total Pages: 620
  • Publisher: Humana Press
• [Remarks] 北里大学医学部分子遺伝学
  • URL: http://www.med.kitasato-u.ac.jp/~molgen/
• [Remarks] 北里大学分子遺伝
  • URL: http://www.med.kitasato-u.ac.jp/~molgen/