In vivo Regulation of the Cell Fates Utilizing a Novel Inducible System of Protein Expression

• Project/Area Number: 21591231
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Hematology
• Research Institution: The University of Tokyo
• Principal Investigator: OTSU Makoto 東京大学, 医科学研究所, 特任准教授 (30361330)
• Co-Investigator(Kenkyū-buntansha): TAKAUCHI Yasuo 北里大学, 医学部, 講師 (60286359)
• Project Period (FY): 2009 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2011: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2010: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2009: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 造血幹細胞 / レトロウイルスベクター / レンチウイルスベクター / 発現調節 / 細胞老化

Research Abstract

As planned, I constructed the inducible retroviral system that could allow rapid and dose-related regulation of expression of the target protein. It, however, turned out that the degradation-domain(DD)/Shield-1 system did have an intrinsic defect in itself, which was compatible with significant expression leak. I thus decided to change gears to the combined use of DD/Shield-1 and tetracycline-inducible system, the latter of which was originally developed by Dr. Bujard. I utilized so-called "All-in-One LV", the lentiviral system that was generated by my colleague to allow containing all the required elements for the system to work within a single cassette. Among the combination tested, I finally figured out that the system comprised of Tet-ON and DD/Shield-1 was the best performer so that it allowed the lowest leak and prompt expression upon dual stimulation. Although the overall progress remain behind the schedule due to the change of plans, I believe that the system is now ready to be tested in vivo, once the inducible target gene is introduced within this newly developed dual-regulation lentiviral vector.

Research Products

• [Journal Article] Frequent pathway mutations of splicing machinery in myelodysplasia (2011)
  • Author(s): Yoshida K, Sanada M, Shiraishi Y, Nowak D, Nagata Y, Yamamoto R, Sato Y, Sato-Otsubo A, Kon A, Nagasaki M, Chalkidis G, Suzuki Y, Shiosaka M, Kawahata R, Yamaguchi T, Otsu M, Obara N, Sakata-Yanagimoto M, Ishiyama K, Mori H, Nolte F, Hofmann WK, Miyawaki S, Sugano S, Haferlach C, Koeffler HP, Shih LY, Haferlach T, Chiba S, Nakauchi H, Miyano S, Ogawa S
  • Journal Title: Nature Volume: 478 Pages: 64-69
  • Peer Reviewed
• [Journal Article] Minimum requirement of donor cells to reduce the glycolipid storage following bone marrow transplantation in a murine model of Fabry disease (2011)
  • Author(s): Yokoi T, Kobayashi H, Shimada Y, Eto Y, Ishige N, Kitagawa T, Otsu M, Nakauchi H, Ida H, Ohashi T
  • Journal Title: J Gene Med Volume: 13 Pages: 262-268
  • Peer Reviewed
• [Journal Article] Intracellular estrogen receptor-binding fragment-associated antigen 9 exerts in vivo tumor-promoting effects via its coiled-coil region (2011)
  • Author(s): Maeyama Y, Otsu M, Kubo S, Yamano T, Iimura Y, Onodera M, Kondo S, Sakiyama Y, Ariga T
  • Journal Title: Int J Oncol Volume: 39 Pages: 41-49
  • Peer Reviewed
• [Journal Article] Heterozygous ITGA2B R995W mutation inducing constitutive activation of the alphaIIbbeta3 receptor affects proplatelet formation and causes congenital macrothrombocytopenia (2011)
  • Author(s): Kunishima S, Kashiwagi H, Otsu M, Takayama N, Eto K, Onodera M, Miyajima Y, Takamatsu Y, Suzumiya J, Matsubara K, Tomiyama Y, Saito H
  • Journal Title: Blood Volume: 117 Pages: 5479-5484
  • Peer Reviewed
• [Journal Article] Growth promotion of genetically modified hematopoietic progenitors using an antibody/c-Mpl chimera (2011)
  • Author(s): Kawahara M, Chen J, Sogo T, Teng J, Otsu M, Onodera M, Nakauchi H, Ueda H, Nagamune T
  • Journal Title: Cytokine Volume: 55 Pages: 402-408
  • Peer Reviewed
• [Journal Article] Minimum requirement of donor cells to reduce the glycolipid storage following bone marrow transplantation in a murine model of Fabry disease (2011)
  • Author(s): Yokoi, T., et al.
  • Journal Title: The journal of gene medicine Volume: 13 Pages: 262-268
  • Peer Reviewed
• [Journal Article] In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling (2011)
  • Author(s): S.Nishimura
  • Journal Title: Blood Volume: 119 Issue: 8 Pages: 128-131
  • DOI: 10.1182/blood-2011-09-381400
  • Peer Reviewed
• [Journal Article] Heterozygous ITGA2B R995W mutation inducing constitutive activation of the {alpha}IIb{beta}3 receptor affects proplatelet formation and causes congenital macrothrombocytopenia (2011)
  • Author(s): Kunishima S, Kashiwagi H, Otsu M, Takayama N, Eto K, Onodera M, Miyajima Y, Takamatsu Y, Suzumiya J, Matsubara K, Tomiyama Y, Saito H
  • Journal Title: Blood Volume: 117 Issue: 20 Pages: 5479-5484
  • DOI: 10.1182/blood-2010-12-323691
  • Peer Reviewed
• [Journal Article] Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells (2010)
  • Author(s): Takayama N, Nishimura S, Nakamura S, Shimizu T, Ohnishi R, Endo H, Yamaguchi T, Otsu M, Nishimura K, Nakanishi M, Sawaguchi A, Nagai R, Takahashi K, Yamanaka S, Nakauchi H, Eto K
  • Journal Title: J Exp Med Volume: 207 Pages: 2817-30
  • Peer Reviewed
• [Journal Article] Reprogramming adult hematopoietic cells (2010)
  • Author(s): Kaneko S, Otsu M, Nakauchi H
  • Journal Title: Curr Opin Hematol Volume: 17 Pages: 271-5
  • Peer Reviewed
• [Journal Article] Reduction of N-glycolylneuraminic acid in human induced pluripotent stem cells generated or cultured under feeder-and serum-free defined conditions (2010)
  • Author(s): Hayashi Y, Chan T, Warashina M, Fukuda M, Ariizumi T, Okabayashi K, Takayama N, Otsu M, Eto K, Furue MK, Michiue T, Ohnuma K, Nakauchi H, Asashima M
  • Journal Title: PLoS One Volume: 5
  • Peer Reviewed
• [Journal Article] Deregulated intracellular signaling by mutated c-CBL in myeloid neoplasms (2010)
  • Author(s): Ogawa S, Shih LY, Suzuki T, Otsu M, Nakauchi H, Koeffler HP, Sanada M
  • Journal Title: Clin Cancer Res Volume: 16 Pages: 3825-31
  • Peer Reviewed
• [Journal Article] Gain-of-function c-CBL mutations associated with uniparental disomy of 11q in myeloid neoplasms (2010)
  • Author(s): Ogawa S, Sanada M, Shih LY, Suzuki T, Otsu M, Nakauchi H, Koeffler HP
  • Journal Title: Cell Cycle Volume: 9 Pages: 1051-6
  • Peer Reviewed
• [Journal Article] Transient activation of c-MYC expression is critical for efficient platelet generation from human induced nlurinotent stem cells. (2010)
  • Author(s): Takayama N, et al.
  • Journal Title: J Exp Med Volume: 207 Pages: 2817-2830
  • Peer Reviewed
• [Journal Article] Reduction of N-glycolylneuraminic acid in human induced pluripotent stem cells generated or cultured under feeder-and serum-free defined conditions. (2010)
  • Author(s): Hayashi Y, et al.
  • Journal Title: PLoS One Volume: 5
  • Peer Reviewed
• [Journal Article] Reprogramming adult hematopoietic cells. (2010)
  • Author(s): Kaneko S, Otsu M, Nakauc hi H
  • Journal Title: Curr Opin Hematol Volume: 17 Pages: 271-275
  • Peer Reviewed
• [Journal Article] Gain-of-function c-CBL mutations associated with uniparental disomy of 11q in myeloid neoplasms. (2010)
  • Author(s): Ogawa S, et al.
  • Journal Title: Cell Cycle Volume: 9 Pages: 1051-1056
  • Peer Reviewed
• [Journal Article] Deregulated intracellular signaling by mutated c-CBL in myeloid neonlasms. (2010)
  • Author(s): Ogawa S, et al.
  • Journal Title: Clin Cancer Res Volume: 16 Pages: 3825-3831
  • Peer Reviewed
• [Journal Article] T cell growth control using hapten-specific antibody/interleukin-2 receptor chimera (2009)
  • Author(s): Sogo T, Kawahara M, Ueda H, Otsu M, Onodera M, Nakauchi H, Nagamune T
  • Journal Title: Cytokine Volume: 46 Pages: 127-136
  • Peer Reviewed
• [Journal Article] Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms (2009)
  • Author(s): Sanada M, Suzuki T, Shih LY, Otsu M, Kato M, Yamazaki S, Tamura A, Honda H, Sakata-Yanagimoto M, Kumano K, Oda H, Yamagata T, Takita J, Gotoh N, Nakazaki K, Kawamata N, Onodera M, Nobuyoshi M, Hayashi Y, Harada H, Kurokawa M, Chiba S, Mori H, Ozawa K, Omine M, Hirai H, Nakauchi H, Koeffler HP, Ogawa S
  • Journal Title: Nature Volume: 460 Pages: 904-8
  • Peer Reviewed
• [Journal Article] Definitive proof for direct reprogramming of hematopoietic cells to pluripotency (2009)
  • Author(s): Okabe M, Otsu M, Ahn DH, Kobayashi T, Wakiyama Y, Morita Y, Onodera M, Eto K, Ema H, Nakauchi H
  • Journal Title: Blood Volume: 114 Pages: 1764-7
  • Peer Reviewed
• [Journal Article] The actin polymerization regulator WAVE2 is required for early bone marrow repopulation by hematopoietic stem cells (2009)
  • Author(s): Ogaeri T, Eto K, Otsu M, Ema H, Nakauchi H
  • Journal Title: Stem Cells Volume: 27 Pages: 1120-1129
  • Peer Reviewed
• [Journal Article] Definitive proof for direct reprogramming of hematopoietic cells to pluripotency. (2009)
  • Author(s): Okabe M, et al.
  • Journal Title: Blood 114 Pages: 1764-1767
  • Peer Reviewed
• [Journal Article] CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity. (2009)
  • Author(s): Nishimura S, et al.
  • Journal Title: Nature Medicine 15 Pages: 914-920
  • Peer Reviewed
• [Journal Article] Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms. (2009)
  • Author(s): Sanada M, et al.
  • Journal Title: Nature 460 Pages: 904-908
  • Peer Reviewed
• [Journal Article] The actin polymerization regulator WAVE2 is required for early bone marrow repopulation by hematopoietic stem cells. (2009)
  • Author(s): Ogaeri T, et al.
  • Journal Title: Stem Cells 27 Pages: 1120-1129
  • Peer Reviewed
• [Presentation] Myeloablationを必要としない造血幹細胞移植法の確立に向けてTowards the establishment of hematopoietic stem cell transplantation without the need for myeloablation (2012)
  • Author(s): Makoto Otsu
  • Organizer: 第34回日本造血細胞移植学会総会・日本再生医療学会合同シンポジウム[造血幹細胞移植の未来]
  • Place of Presentation: 大阪
  • Year and Date: 2012-02-24
• [Presentation] Towards the establishment of hematopoietic stem cell transplantation without the need for myeloablation (2012)
  • Author(s): Makoto Otsu
  • Organizer: 第34回日本造血細胞移植学会総会日本再生医療学会合同シンポジウム
  • Place of Presentation: Osaka, Japan(招待講演)
  • Year and Date: 2012-02-24
• [Presentation] Stem Cell Gene Therapy for Primary Immunodeficiency Diseases.-Where are we now and where should we go?- (2011)
  • Author(s): Makoto Otsu
  • Organizer: 第17回日本遺伝子治療学会シンポジウム
  • Place of Presentation: 福岡
  • Year and Date: 2011-07-17
• [Presentation] STEM CELL GENE THERAPY FOR PRIMARY IMMUNODEFICIENCY DISEASES : WHERE ARE WE TODAY AND WHERE SHOULD WE GO? (2011)
  • Author(s): Makoto Otsu
  • Organizer: JSGT 17th Annual Meeting
  • Place of Presentation: Fukuoka, Japan(招待講演)
  • Year and Date: 2011-07-17
• [Presentation] より良い造血細胞移植を目指して:造血幹細胞における生着および骨髄再構築能の制御 (2011)
  • Author(s): Makoto Otsu
  • Organizer: 第10回神奈川小児血液・感染症フォーラム
  • Place of Presentation: 横浜
  • Year and Date: 2011-02-09
• [Presentation] Evidence for the Involvement of CXCR4 Signaling in in vivo Self-Renewal of Transplanted Hematopoietic Stem Cells (2011)
  • Author(s): CHEN-YI LAI, et al.
  • Organizer: The 14th Annual Meeting of the American Society of Gene Therapy
  • Place of Presentation: Seattle, WA, USA
• [Presentation] Patient Autologous iPSCs in the Modelling and Treatment of Chronic Granulomatous Disease (2011)
  • Author(s): LIN HUAN-TING, et al.
  • Organizer: The 18th Annual Congress of the European Society of Gene Therapy
  • Place of Presentation: Brighton, UK
• [Presentation] Irradiated bone marrow environment impairs hematopoietic stem cells by exposure to tumor necrosis factor a (2010)
  • Author(s): Suzuki, M, et al.
  • Organizer: 52^<nd> ASH annual meeting
  • Place of Presentation: Orlando, FL, USA
  • Year and Date: 2010-12-04
• [Presentation] Update on a Japanese clinical trial of Stem Cell Gene Therapy for ADA-Deficiency (2010)
  • Author(s): Otsu, M, et al.
  • Organizer: 18th annual congress of the ESGCT
  • Place of Presentation: Milan, Italy
  • Year and Date: 2010-10-22
• [Presentation] Role of CXCR4 Signaling in Hematopoietic Stem Cell Repopulation (2010)
  • Author(s): Lai, CY, et al.
  • Organizer: 16^<th> annual meeting of JSGT
  • Place of Presentation: Tochigi-ken General Culture Center
  • Year and Date: 2010-07-01
• [Presentation] Characterization of iPS cells generated from murine hematopoietic cells (2010)
  • Author(s): Okabe, M, et al.
  • Organizer: 16^<th> annual meeting of JSGT
  • Place of Presentation: Tochigi-ken General Culture Center
  • Year and Date: 2010-07-01
• [Presentation] Stem Cell Gene Therapy For Adenosine Deaminase-Deficiency : A Report of Six-Year Outcomes In 2 Treated Patients (2010)
  • Author(s): Otsu, M, et al.
  • Organizer: 16^<th> annual meeting of JSGT
  • Place of Presentation: Tochigi-ken General Culture Center
  • Year and Date: 2010-07-01
• [Presentation] Update of Stem Cell Gene Therapy Clinical Trial for ADA-Deficiency in Japan (2010)
  • Author(s): Otsu, M, et al.
  • Organizer: 13th annual meeting of ASGCT
  • Place of Presentation: Washington DC, USA
  • Year and Date: 2010-05-22
• [Presentation] Hematopoietic Cells and Induced Pluripotent Stem Cells : Good Friends in Regenerative Medicine? (2010)
  • Author(s): 大津真
  • Organizer: 日本再生医療学会総会
  • Place of Presentation: 広島
  • Year and Date: 2010-03-18
• [Presentation] 先天性免疫不全症の遺伝子細胞治療 (2009)
  • Author(s): 大津真
  • Organizer: 第37回日本臨床免疫学会総会
  • Place of Presentation: 東京
  • Year and Date: 2009-11-14
• [Presentation] Definitive proof for direct reprogramming of hematopoietic cells to pluripotency (2009)
  • Author(s): Okabe M, et al.
  • Organizer: ISSCR 7^<th> Annual Meeting
  • Place of Presentation: Barcelona, Spain
• [Presentation] A threshold in expression levels of gp91phox transgene limits the degree of functional correction in granulocytes after gene therapy for CGD. (2009)
  • Author(s): 竹内康雄, 他
  • Organizer: The 17^<th> Annual Congress of the ESGCT
  • Place of Presentation: Hannover, Germany
• [Presentation] 造血幹細胞移植において放射線照射された骨髄環境が造血幹細胞の能力に影響を与える. (2009)
  • Author(s): 鈴木幸恵, 他
  • Organizer: 第71回日本血液学会学術集会
  • Place of Presentation: 京都
• [Presentation] 抗CD40Lモノクローナル抗体投与を用いた骨髄混合キメリズム誘導による慢性肉芽腫症モデルマウスの治療 (2009)
  • Author(s): 竹内康雄, 他
  • Organizer: 第71回日本血液学会学術集会
  • Place of Presentation: 京都
• [Book] 先天性免疫不全症の遺伝子細胞治療 (2010)
• [Remarks]
  • URL: http://stemcell-u-tokyo.org/scb/
• [Remarks]
  • URL: http://stemcell-u-tokyo.org/sct/
• [Remarks]
  • URL: http://stemcell-u-tokyo.org/sct/
• [Patent(Industrial Property Rights)] 誘導型多能性幹細胞の製造方法 (2011)
  • Inventor(s): 中内啓光,大津真,高山直也,安東赫
  • Industrial Property Rights Holder: 東京大学
  • Filing Date: 2011-06-15
  • Overseas