1999 Fiscal Year Annual Research Report

AAVを利用した染色体部位特異的遺伝子組込み法の開発とその造血系細胞への応用

Research Project

Project/Area Number	10470213
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)	卜部匡司自治医科大学, 医学部, 助手 (40213516) 久米晃啓自治医科大学, 医学部, 講師 (10264293)
Keywords	遺伝子治療 / 遺伝子導入 / 遺伝子組込み / AAV / AAVS1領域 / Rep / ITR / TVI法
Research Abstract	アデノ随伴ウイルス(AAV)のITRとRepという二つのコンポーネントを利用した第19番染色体部位特異的遺伝子組込み法[TVI(Targeted Vector Integration)法]の開発、特に、Rep蛋白質の機能解析と細胞毒性の制御法に焦点を当てた検討を行った。本年度は、これまでに開発した多数の変異Rep発現ベクターの機能解析をさらに詳細に行った。その他、ランダムに変異を挿入したRep遺伝子の解析も進めた。具体的には、AAVS1の塩基配列を持つエピソーム型ベクター(EBV由来のEBNA1とori配列を持つ)を予め293細胞に導入しておき、その系で、AAVS1特異的組込み活性を保持し、細胞毒性の低くなった変異Rep遺伝子をスクリーニングする作業を進めた。このストラテジーで得られた変異Rep遺伝子に関しては、現在確認実験を行っている。また、TVI法を行った場所の染色体組込み部位については、AAVS1以外の未同定の部位も含まれる。そこで、遺伝子組込み部位を明らかにするため、Alu-PCR法による解析を行った。検討した範囲では、AAVS1以外の組込み部位に共通の特徴的な塩基配列はなかったが、K562細胞から得られたDNAの一つにGAGTの繰り返し配列を認めた。また、組込みが起こった部位のオリジナルの塩基配列を今後解析することにり、TVI法による遺伝子組込み機構に関して有用な情報が得られるものと考えられる。

Research Products

(12 results)

All Other

All Publications (12 results)

[Publications] Matsuda,K.M.: "A novel strategy for the tumor angiogenesis-targeted gene therapy : Generation of angiostatin endogenous plasminogen by protease gene transfer"Cancer Gene Ther.. (in press).
[Publications] Urabe,M.: "DNA/calcium phosphate precipitates mixed with medium are stable and maintain high transduction efficiency"Anal.Biochem.. 278. 91-92 (2000)
[Publications] Maeda,Y.: "Endogenously generated nitric oxide by nitric-oxide synthase gene transfer inhibits cellular proliferation"J.Phamacol.Exp.Therapeut.. 292. 387-393 (2000)
[Publications] Ogasawara,Y.: "Potential application of dominant negative retinoic acid receptor genes for ex vivo expansion of hematopoietic stem cells"Gene Ther.Mol.Biol.. 3. 293-300 (1999)
[Publications] Ogasawara,Y.: "Highly-regulated expression of adeno-associated virus large Rep proteins in stable 293 cell lines using the Cre-loxP switching system"J.Gen.Virol.. 80. 2477-2480 (1999)
[Publications] Xu,R.: "A selective amplifier gene for tamoxifen-inducible expansion of hematopoietic cells"J.Gene Med.. 1. 236-244 (1999)
[Publications] Matsuda,K.M.: "Development of a modified selective a mplifier gene for hematopoietic stem cell gene therapy"Gene Ther.. 6. 1038-1044 (1999)
[Publications] Kume,A.: "Green fluorescent protein as a selectable marker of retrovirally transduced hematopoietic progenitors"Stem Cells. 17. 226-232 (1999)
[Publications] Kume,A.: "A G-CSF receptor-gyrase B fusion gene : a new type of molecular switch for expansion of genetically modified hematopoietic cells"Biochem.Biophys.Res.Commun.. 260. 9-12 (1999)
[Publications] Ozawa,K.: "Strategies for gene therapy of Parkinson's disease using adeno-associated virus(AAV) vectors"Biogenic Amines. 15. 21-37 (1999)
[Publications] Ogasawara,Y.: "Efficient production of adeno-associated virus vectors using split-type helper plasmids"Jpn.J.Cancer Res.. 90. 476-483 (1999)
[Publications] Urabe,M.: "Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep 78 protein"J.Virol.. 73. 2682-2693 (1999)

1999 Fiscal Year Annual Research Report

AAVを利用した染色体部位特異的遺伝子組込み法の開発とその造血系細胞への応用

Principal Investigator

小澤 敬也 自治医科大学, 医学部, 教授 (30137707)

Research Products

[Publications] Matsuda,K.M.: "A novel strategy for the tumor angiogenesis-targeted gene therapy : Generation of angiostatin endogenous plasminogen by protease gene transfer"Cancer Gene Ther.. (in press).

[Publications] Urabe,M.: "DNA/calcium phosphate precipitates mixed with medium are stable and maintain high transduction efficiency"Anal.Biochem.. 278. 91-92 (2000)

[Publications] Maeda,Y.: "Endogenously generated nitric oxide by nitric-oxide synthase gene transfer inhibits cellular proliferation"J.Phamacol.Exp.Therapeut.. 292. 387-393 (2000)

[Publications] Ogasawara,Y.: "Potential application of dominant negative retinoic acid receptor genes for ex vivo expansion of hematopoietic stem cells"Gene Ther.Mol.Biol.. 3. 293-300 (1999)

[Publications] Ogasawara,Y.: "Highly-regulated expression of adeno-associated virus large Rep proteins in stable 293 cell lines using the Cre-loxP switching system"J.Gen.Virol.. 80. 2477-2480 (1999)

[Publications] Xu,R.: "A selective amplifier gene for tamoxifen-inducible expansion of hematopoietic cells"J.Gene Med.. 1. 236-244 (1999)

[Publications] Matsuda,K.M.: "Development of a modified selective a mplifier gene for hematopoietic stem cell gene therapy"Gene Ther.. 6. 1038-1044 (1999)

[Publications] Kume,A.: "Green fluorescent protein as a selectable marker of retrovirally transduced hematopoietic progenitors"Stem Cells. 17. 226-232 (1999)

[Publications] Kume,A.: "A G-CSF receptor-gyrase B fusion gene : a new type of molecular switch for expansion of genetically modified hematopoietic cells"Biochem.Biophys.Res.Commun.. 260. 9-12 (1999)

[Publications] Ozawa,K.: "Strategies for gene therapy of Parkinson's disease using adeno-associated virus(AAV) vectors"Biogenic Amines. 15. 21-37 (1999)

[Publications] Ogasawara,Y.: "Efficient production of adeno-associated virus vectors using split-type helper plasmids"Jpn.J.Cancer Res.. 90. 476-483 (1999)

[Publications] Urabe,M.: "Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep 78 protein"J.Virol.. 73. 2682-2693 (1999)

小澤敬也自治医科大学, 医学部, 教授 (30137707)