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2001 Fiscal Year Final Research Report Summary

Fundamental researches toward gene therapy of Hemophilia A utilizing adeno-associated virus vectors

Research Project

Project/Area Number 12671003
Research Category

Grant-in-Aid for Scientific Research (C)

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

Principal Investigator

MIZUKAMI Hiroaki  Jichi Medical School, Dept. Medicine, Assis. Prof., 医学部, 助手 (20311938)

Co-Investigator(Kenkyū-buntansha) MADOIWA Seiji  Jichi Medical School, Dept. Medicine, Assis. Prof., 医学部, 講師 (70296119)
Project Period (FY) 2000 – 2001
Keywordsadeno-associated virus / AAV vector / Hemophilia A / Coagnlation Factor VIII / promoters / serotype / skeletal muscle / liver
Research Abstract

In this study we determined the optimal conditions for the transaction utilizing AAV vectors prior to the construction of vectors for FVIII. For this purpose, we investigated the amount of murine erythropoietin (Epo) within systemic circulation by comparing different serotypes as well as promoters. For the muscle transduction, we compared the difference by serotypes with cytomegalovirus-derived (CMV) promoter. Each mouse received a total of 6 x 10^<10> genome copies of each serotype vector into bilateral gastrocunemius muscle. For the muscle-mediated expression, serotype 1 showed the highest Epo concentration, followed by 5, 4, 3 and 2 at 4 weeks. For the liver-mediated transduction, CMV, CAG, EF-1α and PGK promoters were compared utilizing AAV serotype 2 platform. Vectors were intraportally administered, with the dose of both 1 x 10^<10>vg and 1 x 10^<11>vg/body As for the promoter analysis in the liver, CAG promoter achieved the highest concentration of Epo, followed by CMV, PGK and EF-1α. For the comparison of serotypes, CAG promoter was used and serotype 5 worked best in the liver. According to these results, we designed the AAV vector structure for the systemic delivery of Factor VIII. For the liver and muscle, the combination of CAG promoter plus AAV5 capsid and CMV promoter with AAV1 structure were utilized to express both heavy and light chains of Factor VIII. The competence of this strategy along with the potency of these plasmids was confirmed following experiments with transfection. Utilizing these plasmids, we prepared the necessary amounts of AAV vectors for in vivo transduction. Also, knockout mice for Factor VIII were obtained. We are now preparing for the injection into the mouse model and pursuing the therapeutic outcome of this strategy. If it works well, we will move to the models of larger animals to validate its utility toward human gene therapy.

  • Research Products

    (32 results)

All Other

All Publications (32 results)

  • [Publications] Hasumi Y, et al.: "Soluble FLT-1 Expression Suppresses Carcinomatous Ascites in Nude Mice Bearing Ovarian Cancer"Cancer Research. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Muramatsu S, et al.: "Behavioral Recovery in a Primate Model of Parkinson's Disease by triple transudation"Human Gene Therapy. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Shimpo M, et al.: "AAV-Mediated VEGF Gene Transter in to Skeletal Muscle Stimulates Angiogenesis and Improves・・・"Cardiovascular Research. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Saga Y, et al.: "Expression of HGF/NK4 in Ovarian Cancer cells Suppresses Intraperitoneal Dissemination"Gene Therapy. 8. 1450-1455 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Xin K, et al.: "A Novel Recombinant AAV Vaccine Induces a Long-term Humoral Immune Response to HIV"Human Gene Therapy. 12. 1047-1061 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [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 Commun.. 285. 891-896 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kanazawa T, et al.: "γ-Rays Enhance rAAV-Mediated Transgene Expression and Cytocidal Effect of AAV-HSVtk/ganciclovir on cancer cells"Cancer Gene Therapy. 8. 99-106 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Okada T et al.: "Development and Characterization of an Antisense Mediated Prepackaging Cell line for AAV Vector"Biochem Biophys Res Commun.. 288. 62-68 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kogure K et al.: "Targetedintegration of foreign DNA into a defined locus on chromosome 19 in K562 cells usingAAV-derived components"Int J of Hematology. 73. 469-475 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Handa A et al.: "AAV-3 based Vectors Transcuce Hematopoietic Cells not Susceptible to Transudation with・・・"J of Gen Virology. 81. 2077-2084 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Shen Y, et al.: "Triple Transduction with AAV Vectors Expressing Tyrosine Hydroxylase, Aromatic-L-Amino Acid Decarboxylase, and GTP Cyclohydrolase I for Gene Therapy of Parkinson's Disease"Human Gene Therapy. 11. 1509-1519 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Shen Y et al.: "Autoantibody against Prothrombin Aberrantly Alters the Proenzyme to facilitate Formation of a complex with its physiological inhibitor antithrombin III without thrombin conversion"Blood. 97. 3783-3789 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Matsuda KM et al.: "A Novel strategy for the tumor angiogenesis-targeted gene therapy: generation of angiostatin from endogenous plasminogen by protease gene transfer"Cancer Gene Therapy. 7. 589-596 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Wong L, et al.: "Delayed delivery of AAV-GDVF Prevents nignal neuroreduction・・・"Gene Therapy. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Okada T, et al.: "Methods in Enzymology.Vol.346"Academic Press. 16 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ozawa K et al.: "Progress in Gene Therapy-basic and clinical"VSP. 18 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hasumi Y, et al: "Soluble Flt- 1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer"Cancer Res.. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Muramatsu S, et al.: "Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adenoassociated viral vectors expressing dopamine synthesizing enzymes"Hum. Gene Ther. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Shimpo M, et al.: "AAV-mediated VEGF gene transfer into skeletal muscle stimulates angiogenesis and improves blood flow in a rat hindlimb ischemia model"Cardiovasc. Res.. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Saga Y, et al: "Expression of HGF/NK4 in ovarian cancer cells suppresses intraperitoneal dissemination and extends host survival"Gene Therapy. 8. 1450-1455 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Xin K-Q, et al.: "A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodeficiency virus"Hum Gene Ther. 12. 1047-1061 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [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 Commun. 285. 891-896 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kanazawa T, et al.: "Gamma rays enhance rAAV-mediated transgene expression and cytocidal effect of AAV-HSVtk/gancyclovir on cancer cells"Cancer Gene Ther. 8. 99-106 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Okada T., et al.: "Development and characterization of an antisense-mediated prepackaging cell line for adeno-associated virus vector production"Biochem Biophys Res Commun. 288. 62-68 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kogure K, et al.: "Targeted Integration of Foreign DNA into a Defined Locus on Chromosome 19 in K562 Cells Using AAV-Derived Components"Int J Hematol. 73. 469-475 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Handa A, et al.: "AAV3 based vectors transduce hematopoietic cells not susceptible with AAV2 based vectors"J Gen Virol. 81l. 2077-2084 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Shen Y, et al.: "Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic 1-amino acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson's Disease"Hum Gene Ther. 11. 1509-1519 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Madoiwa S, et al.: "Autoantibody against prothrombin aberrantly alters the proenzyme to facilitate formation of a complex with its physiological inhibitor antithrombin III without thrombin conversion"Blood. 97(12). 3783-3789 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Matsuda KM, et.al: "A novel strategy for the tumor angiogenesis-targeted gene therapy : generation of angiostain from endogenous plasminogen by protease gene transfer"Cancer Gene Ther. 7(4). 589-596 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Wang L, et al: "Delayed delivery of AAV-GDNF prevents nigral neuroreduction and promotes functional recovery in a rat model of Parkinson' s disease"Gene Therapy. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Okada T, et al: "AAV mediated gene transfer for gene therapy of ischemia-induced neuronal death"Methods in Enzymology. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ozawa K, et al: "Adeno-associated virus vecors and gene therapy of Parkinson' s disease"Progress in Gene Therapy - Basic and Clinical Frontiers. 289-306 (2000)

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

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Published: 2003-09-17  

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