2001 Fiscal Year Annual Research Report

アデノ随伴ウイルス(AAV)を利用した遺伝子治療テクノロジーの開発研究

Research Project

Project/Area Number	12470203
Research Institution	Jichi Medical School
Principal Investigator	小澤敬也自治医科大学, 医学部, 教授 (30137707)
Co-Investigator(Kenkyū-buntansha)	水上浩明自治医科大学, 医学部, 助手 (20311938) 久米晃啓自治医科大学, 医学部, 助教授 (10264293)
Keywords	遺伝子治療 / 遺伝子導入 / AAVベクター / パッケージング細胞株 / AAVS1領域 / Rep / ITR / TVI法
Research Abstract	アデノ随伴ウイルス(AAV)を利用した遺伝子導入法の本格的実用化を目指し、以下の研究を行った。 1.AAVベクター作製法の開発:細胞毒性を有するAAV蛋白質の発現を制御したAAVベクター作製用パッケージング細胞株の開発を行った。即ち、RepおよびCapに対するアンチセンスmRNAを恒常的に発現させてRepおよびCapの発現を阻害し、必要時にCre-loxP法によってアンチセンスを抑え、Rep/Cap蛋白質の発現を誘導する新規システムである。得られた細胞株におけるAAVベクターのパッケージング機能を確認するため、LacZ発現AAVベクターとともにAdexCre(moi=1)を感染させ、AAVベクターの産生を確認した。さらにウイルス産生量がCapの発現に依存することに着目し、Capの発現を補うアデノウイルスAVC2Capを共感染させ、作製効率の改善が得られることを見いだした。 2.AAVベクターで導入した遺伝子の高感度検出法の確立と応用:AAVベクターを筋注した動物の種々の臓器・組織における導入遺伝子の広がりを高感度検出法で検討するため、広範なサンプリングを行った。解析は進行中である。 3.AAVのコンポーネント(ITR配列とRep遺伝子)を利用した第19番染色体部位特異的遺伝子組込み(TVI)法の開発:(1)AAV-ITRを連結したマーカー遺伝子とRep発現プラスミドベクターをトランスフェクションした293細胞およびK562細胞を用いて、Alu-PCR法による遺伝子組込み部位近傍の塩基配列解析を引き続き検討した。これまでのところ、AAVS1以外に特徴的な塩基配列は見いだされなかった。(2)Repを一過性に発現させる新しい方法の開発に着手した。

Research Products

(16 results)

All Other

All Publications (16 results)

[Publications] Hasumi, Y.: "Soluble Flt-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer"Cancer Res.. (in press).
[Publications] Wang, L.: "Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease"Gene Ther.. (in press).
[Publications] Shimpo, M.: "AAV-mediated VEGF gene transfer into skeletal muscle stimulates angiogenesis and improves blood flow in a rat hindlimb ischemia model"Cardiovasc. Res.. (in press).
[Publications] Muramatsu, S.: "Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes"Hum.Gene Ther.. (in press).
[Publications] Okada, T.: "Adeno-associated viral vector-mediated gene therapy of ischemia-induced neuronal death"Method.Enzymol.. 346. 378-393 (2002)
[Publications] Kawada, T.: "Rescue of hereditary form dilated cardiomyopathy by rAAV-mediated somaticgene therapy : Amelioration of morphological findings, sarcolemmal permeability, cardiac performances, and the prognosis of TO-2 hamsters"Proc.Natl.Acad.Sci.U.S.A.. 99. 901-906 (2002)
[Publications] Okada, T.: "Development and characterization of an antisense-mediated regulation system for adeno-associated virus vector production with introduction of Cre recombinase"Biochem.Biophys.Res.Commun.. 288. 62-68 (2001)
[Publications] Saga, Y.: "Expression of HGF/NK4 in ovarian cancer cells suppresses intraperitoneal dissemination and extends host survival"Gene Ther.. 8. 1450-1455 (2001)
[Publications] Muramatsu, M.: "Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells"Biochem.Biophys.Res.Commun.. 27. 891-896 (2001)
[Publications] Maeda, Y.: "Adeno-associated virus-mediated transfer of endothelial nitric oxide synthase gene inhibits protein synthesis of rat ventricular cardiomyocytes"Cardiovas. Drug Ther.. 15. 19-24 (2001)
[Publications] Kogure, K.: "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)
[Publications] Xin, K.-Q.: "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)
[Publications] Maeda, Y.: "Adeno-associated virus-mediated transfer of endothelial nitric oxide synthase gene reduces vasoconstrictive response"Exp.Clin.Cardiol.. 6. 50-55 (2001)
[Publications] Kawada, T.: "Morphological and physiological restorations of hereditary form of dilated cardiomyopathy by somatic gene therapy"Biochem.Biophys.Res.Commun.. 284. 431-435 (2001)
[Publications] Kanazawa, T.: "γ-Rays enhance rAAV-mediated transgene expression and cytocidal effect of AAV-HSVtk/ganciclovir on cancer cells"Cancer Gene Ther.. 8. 99-106 (2001)
[Publications] Fan, D.: "Adeno-associated virus vector-mediated triple gene transfer of dopamine synthetic enzymes"Chin.Med.J.. 114. 1276-1279 (2001)

2001 Fiscal Year Annual Research Report

アデノ随伴ウイルス(AAV)を利用した遺伝子治療テクノロジーの開発研究

Principal Investigator

小澤 敬也 自治医科大学, 医学部, 教授 (30137707)

Research Products

[Publications] Hasumi, Y.: "Soluble Flt-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer"Cancer Res.. (in press).

[Publications] Wang, L.: "Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease"Gene Ther.. (in press).

[Publications] Shimpo, M.: "AAV-mediated VEGF gene transfer into skeletal muscle stimulates angiogenesis and improves blood flow in a rat hindlimb ischemia model"Cardiovasc. Res.. (in press).

[Publications] Muramatsu, S.: "Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes"Hum.Gene Ther.. (in press).

[Publications] Okada, T.: "Adeno-associated viral vector-mediated gene therapy of ischemia-induced neuronal death"Method.Enzymol.. 346. 378-393 (2002)

[Publications] Kawada, T.: "Rescue of hereditary form dilated cardiomyopathy by rAAV-mediated somaticgene therapy : Amelioration of morphological findings, sarcolemmal permeability, cardiac performances, and the prognosis of TO-2 hamsters"Proc.Natl.Acad.Sci.U.S.A.. 99. 901-906 (2002)

[Publications] Okada, T.: "Development and characterization of an antisense-mediated regulation system for adeno-associated virus vector production with introduction of Cre recombinase"Biochem.Biophys.Res.Commun.. 288. 62-68 (2001)

[Publications] Saga, Y.: "Expression of HGF/NK4 in ovarian cancer cells suppresses intraperitoneal dissemination and extends host survival"Gene Ther.. 8. 1450-1455 (2001)

[Publications] Muramatsu, M.: "Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells"Biochem.Biophys.Res.Commun.. 27. 891-896 (2001)

[Publications] Maeda, Y.: "Adeno-associated virus-mediated transfer of endothelial nitric oxide synthase gene inhibits protein synthesis of rat ventricular cardiomyocytes"Cardiovas. Drug Ther.. 15. 19-24 (2001)

[Publications] Kogure, K.: "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)

[Publications] Xin, K.-Q.: "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)

[Publications] Maeda, Y.: "Adeno-associated virus-mediated transfer of endothelial nitric oxide synthase gene reduces vasoconstrictive response"Exp.Clin.Cardiol.. 6. 50-55 (2001)

[Publications] Kawada, T.: "Morphological and physiological restorations of hereditary form of dilated cardiomyopathy by somatic gene therapy"Biochem.Biophys.Res.Commun.. 284. 431-435 (2001)

[Publications] Kanazawa, T.: "γ-Rays enhance rAAV-mediated transgene expression and cytocidal effect of AAV-HSVtk/ganciclovir on cancer cells"Cancer Gene Ther.. 8. 99-106 (2001)

[Publications] Fan, D.: "Adeno-associated virus vector-mediated triple gene transfer of dopamine synthetic enzymes"Chin.Med.J.. 114. 1276-1279 (2001)

小澤敬也自治医科大学, 医学部, 教授 (30137707)