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1997 Fiscal Year Final Research Report Summary

Development of a novel gene therapy technology for site-specific integration of large-sized genes

Research Project

Project/Area Number 08457280
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Hematology
Research InstitutionJichi Medical School

Principal Investigator

OZAWA Keiya  Jichi Medical School, Faculty of Medicine, Professor, 医学部, 教授 (30137707)

Co-Investigator(Kenkyū-buntansha) URABE Masashi  Jichi Med.Sch., Faculty of Medicine, Research Associate, 医学部, 助手 (40213516)
MANO Hiroyuki  Jichi Med.Sch., Faculty of Medicine, Associate Professor, 医学部, 助教授 (90240704)
Project Period (FY) 1996 – 1997
KeywordsGene therapy / Gene transfer / integration / AAV / AVVS1 locus / Rep / ITR / TVI method
Research Abstract

The desirable gene-delivery system for gene therapy should have several features, including 1) integration of the delivered gene in a predictable or site-specific manner into the target genome, 2) long-term expression of the transgene, and 3) the ability to deliver DNA sequences of sufficiently large size to include all the regulatory elements. The development of a novel gene delivery system, termed targeted vector integration (TVI), is currently in progress to fulfilll the above prerequisites. The system is based on adeno-associated virus (AAV) which preferentially integrates into the human genome at a defined locus, called AAVI1, on chromosome 19 (19q13.3-qter). The AAV-Rep proteins are considered to mediate integration of DNA sequence containing AAV-ITR (inverted terminal repeat) into AAVS1 locus, through the formation of a complex between GAGC repeats within ITR and a similar sequence in AAVS1 locus. There are 4 different Rep proteins (Rep78, Rep68, Rep52, and Rep40). Aiming at determining the Rep (s), which confer the ability of site-specific integration of ITR-linked genes, we constructed various plasmids that express individual Rep proteins. These plasmids were co-transfected into 293 cells with the plasmid containing a LacZ expression cassette flanked by ITRs. A PCR-based dot blot assay, Southern analysis, and FISH analysis were conducted to detect site-specific integration. The results showed that the large Rep (78 ot 68) is necessary for the site-specific integration of ITR-linked genes. In addition, mutant Rep proteins with R107A,K136A,or R138A showed the decreased binding to GAGC repeat and no nicking activity. These mutants also lost the site-specific integration activity. The present study will be valuable to develop the more refined TVI system.

  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] Yoji Ogasawara: "The use of heterologous promoters for adeno-associated virus(AAV)protein expression in AAV vector production" Microbiol.Immunol.(in press). (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Masaki Kokubun: "Apoptosis-mediated regulation of recombinant human granulocyte colony-stimulating factor production by genetically engineered fibroblads" Gene Therapy. (in press). (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Richard T Surosky: "Adeno-associated virus Rep proteins target DNA seguences to a unique locus in the human genome" J.Virol.71. 7951-9751 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Keiko Ito: "Development of a novel selective amplifier gene for controllable expansion of transduced hematopoietic cells" Blood. 90. 3884-3892 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Masashi Urabe: "A novel dicistronic AAV(adeno-associated virus)Vector using a short IRES(internal tibosome entry site) segment derived from hepatitis C virus genome" Gene. 200. 157-162 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yoshikazu Maeda: "Gene transfer into vascular cells using adeno-associated virus(AAV)vectors" Cardiovascular Res.35. 514-521 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ogasawara, Y., Urabe, M., and Ozawa, K.: "The use of heterologous promoters for adeno-associated virus (AAV) protein expression in AAV vector production." Microbiol.Immunol.(in press). (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kokubun, M., Kume, A., Urabe, M., Mano, H., Kakizuka, A., Okubo, M., Kasukawa, R., and Ozawa, K.: "Apoptosis-mediated regulation of recombinant human granulocyte colony-stimulating factor production by genetically engineered fibroblasts." Gene Therapy. (in press). (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Surosky, R.T., Urabe, M., Godwin, S.G., McQuinston, S.A., Kurtzman, G.J., Ozawa, K., and Natsoulis, G.: "Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome." J.Virol.71. 7951-7959 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ito, K., Ueda, Y., Kokubun, M., Urabe, M., Inaba, T., Mano, H., Hamada, H., Kitamura, T., Mizoguchi, H., Sakata, T., Hasegawa, M., and Ozawa, K.: "Development of a novel selective amplifier gene for controllable expansion of transduced hematopoietic cells." Blood. 90. 3884-3892 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Urabe, M., Hasumi, Y., Ogasawara, Y., Matsushita, T., Kamoshita, N., Nomoto, A., Colosi, P., Kurtzman, G.J., Tobita, K., and Ozawa, K.: "A novel dicistronic AAV (adeno-associated virus) vector using a short IRES (internal ribosome entry site) segment derived from hepatitis C virus genome." Gene. 200. 157-162 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Maeda, Y., Ikeda, U., Ogasawara, Y., Urabe, M., Takizawa, T., Saito, T., Colosi, P., Kurtzman, G., Shimada, K., and Ozawa, K.: "Gene transfer into vescular cells using adeno-associated virus (AAV) vectors." Cardiovascular Res.35. 514-521 (1997)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-03-16  

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