腫瘍選択的増幅型ハイブリッドウイルスベクターの開発とその制御機構の解明

• Project/Area Number: 13218116
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Jichi Medical University
• Principal Investigator: 岡田 尚已 (岡田 尚巳) 自治医科大学, 医学部, 講師 (00326828)
• Project Period (FY): 2001 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥24,200,000 (Direct Cost: ¥24,200,000); Fiscal Year 2004: ¥6,000,000 (Direct Cost: ¥6,000,000); Fiscal Year 2003: ¥6,100,000 (Direct Cost: ¥6,100,000); Fiscal Year 2002: ¥6,000,000 (Direct Cost: ¥6,000,000); Fiscal Year 2001: ¥6,100,000 (Direct Cost: ¥6,100,000)
• Keywords: がん / 自殺遺伝子治療 / ウイルスベクター / ハイブリッドベクター / アデノウイルス / レトロウイルス / アデノ随伴ウイルス / 遺伝子治療 / 遺伝子発現制御 / ハイブリッドウイルスベクター / Cre-loxP / 幹細胞

Research Abstract

癌の遺伝子治療において様々なベクター系が用いられているが、より高い臨床的効果を期待するために、さらなる改良が求められている。腫瘍内で治療遺伝子を増幅させ遺伝子発現増強とベクターの拡散を促すシステムの実用化をめざし、ハイブリッドベクターの開発とアデノウイルス初期遺伝子群の機能解析を行うことを目的とした。
本年度は、引き続きアデノウイルス初期遺伝子群E1、E2、E4、およびVAの機能を応用し、細胞内で治療ベクターの複製を誘導するシステムを推進した。アデノウイルス初期遺伝子群発現ベクターが生体内でサイレンシングを受けにくくするために、MSCV-LTRの抑制性転写因子結合領域欠失レトロウイルスベクターを応用した。HSV-放発現治療ベクターAAVTk、E1B55k欠失E1遺伝子発現ベクターAAVE1、E1B55k遺伝子発現ベクターAAVE1B55、E2遺伝子発現レトロウイルスベクターDNE2、E4-VA遺伝子発現レトロウイルスベクターDNE4VA、およびRep/Cap発現レトロウイルスベクターDNRepCapの混合感染を腫瘍細胞の系で行った。治療遺伝子として搭載されたHSV-tk遺伝子のコピー数を測定し、ベクターの複製効率と感染条件を検討した。さらに、腫瘍細胞における遺伝子発現を向上させてベクターの複製効率を増強させるため、ベクター感染時にHDAC阻害剤を併用し、遺伝子発現増強効果やその作用機構を解析した。

Research Products

• [Journal Article] Regulation of Osteoclast Apoptosis by Ubiquitination of Proapoptotic BH3-only Bcl-2 Family Member Bim. (2005)
  • Author(s): Akiyama, T., et al.
  • Journal Title: EMBO J. 22・24 Pages: 6653-6664
• [Journal Article] In situ generation of pseudotyped retroviral progeny by adenovirus-mediated transduction of tumor cells enhances the killing effect of HSV-tk suicide gene therapy in vitro and in vivo. (2004)
  • Author(s): Okada T., et al.
  • Journal Title: J.Gene Med. 6・3 Pages: 288-299
• [Journal Article] The Adenovirus E1A and E1B19K genes provide a helper function for transfection-based AAV vector production. (2004)
  • Author(s): Matsushita, T., et al.
  • Journal Title: J.Gen.Virol. 85・Pt8 Pages: 2209-2214
• [Journal Article] A soluble CAR-SCF fusion protein improves adenoviral vector-mediated gene transfer to c-Kit-positive hematopoietic cells. (2003)
  • Author(s): Itoh, A., et al.
  • Journal Title: J.Gene Med. 5・11 Pages: 929-940
• [Journal Article] Interleukin-10-mediated inhibition of angiogenesis and tumor growth in mice bearing VEGF-producing ovarian cancer (2003)
  • Author(s): Kohno, T., et al.
  • Journal Title: Cancer Res. 63・16 Pages: 5091-5094
• [Journal Article] Enhancement of thymidine kinase-mediated killing of malignant glioma by BimS, a BH3-only cell death activator (2003)
  • Author(s): Yamaguchi, T., et al.
  • Journal Title: Gene Ther. 10・5 Pages: 375-385
• [Book] Adeno-associated viral vector-mediated gene therapy of ischemia-induced neuronal death (ed.by M.Ian Phillips) (2002)
  • Author(s): Okada, T., et al.
  • Publisher: Academic Press
• [Patent(Industrial Property Rights)] 遺伝子導入効率増強剤 (2003)
  • Inventor(s): 岡田 尚巳, 小澤 敬也
  • Industrial Property Rights Holder: 藤沢薬品
  • Industrial Property Number: 2003-122968
  • Filing Date: 2003-04-25
• [Patent(Industrial Property Rights)] アデノウイルス吸着架橋剤 (2002)
  • Inventor(s): 伊藤 章, 花園 裕, 岡田 尚巳, 小沢 敬也
  • Industrial Property Rights Holder: 旭化成
  • Industrial Property Number: 2002-299062
  • Filing Date: 2002-10-11
  • Acquisition Date: 2003-07-09
• [Publications] Okada T, et al.: "In situ generation of pseudotyped retroviral progeny by adenovirus-mediated transduction of tumer cells enhances the killing effect of HSV-tk suicide gene therapy in vitro and in vivo"J.Gene Med.. (in press).
• [Publications] Akiyama T, et al.: "Regulation of Osteoclast Apoptosis by Ubiquitination of Proapoptotic BH3-only Bcl-2Family Member Bim."EMBO J.. (in press).
• [Publications] Yamaguchi, et al.: "Enhancement of thymidine kinase-mediated killing of malignant glioma by BimS, a BH3-only cell death activator"Gene Ther.. 10・5. 375-385 (2003)
• [Publications] Kohno T, et al.: "Interleukin-10-mediated inhibition of angiogenesis and tumor growth in mice bearing VEGR-producing ovarian cancer"Cancer Res.. 63・16. 5091-5094 (2003)
• [Publications] Kanazawa T, et al.: "Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice"Gene Ther.. 10・1. 51-58 (2003)
• [Publications] Kametaka M, et al.: "Reduction of CTLL-2 cytotoxicity by induction of apoptosis with a Fas-estrogen receptor chimera"Cancer Sci.. 94・7. 639-643 (2003)
• [Publications] Okada, T. et al.: "Adeno-associated viral vector-mediated gene therapy of ischemia-induced neuronal death"Methods Ehzymol.. 346. 378-393 (2002)
• [Publications] Okada, T. et al.: "Adeno-associated virus vectors for gene transfer to the brain"Methods. 28・2. 237-247 (2002)
• [Publications] Wang, L.J.et al.: "Neuroprotective effects of glial cell line-derived neurotrophic factor mediated by an adeno-associated virus vector in a transgenic animal model of amyotrophic lateral sclerosis"J.Neurosci.. 22・16. 6920-6928 (2002)
• [Publications] Kanazawa, T. et al.: "Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice"Gene Ther.. 10・1. 51-58 (2003)
• [Publications] Yamaguchi, T. et al.: "Enhancement of thymidine kinase-mediated killing of malignant glioma by BimS, a BH3-only cell death activator"Gene Ther.. (in press). (2003)
• [Publications] Nomoto, T. et al.: "Distinct patterns of gene transfer to gerbil hippocampus with recombinant adeno-associated virus type 2 and 5"Neurosci.Lett.. (in press). (2003)
• [Publications] Okada, T., et.al.: "AAV mediated gene transfer for gene therapy of ischemia-induced neuronal death"Method.Enzymol.. 346. 378-393 (2002)
• [Publications] Okada, T., et.al.: "Development and characterization of an antisense-mediated regulation system for adeno-associated virus vector production with introduction of Cre recombinase"Biochem.Biophys.Res.Comm.. 288. 62-68 (2001)
• [Publications] Okada, T., et.al.: "AV.TK-mediated killing of subcutaneous tumors in situ results in effective immunization against established secondary intracranial tumor deposits"Gene Ther.. 8. 1315-1322 (2001)
• [Publications] Muramatsu, M., et.al.: "Reversible Integration of the Dominant Negative Retinoid Receptor Gene for ex Vivo Expansion of Hematopoietic Stem/Progenitor Cells"Biochem.Biophys.Res.Comm.. 285. 891-896 (2001)
• [Publications] Kanazawa, T., et.al.: "Gamma-rays enhance rAAV-mediated transgene expression and cytocidal effect of AAV-HSVtk/ganciclovir on cancer cells"Cancer Gene Ther.. 8. 99-106 (2001)
• [Publications] Wang L., et.al.: "Delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease"Gene Ther.. (in press).