1998 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 University
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 / ITR / Rep / AAVS1領域 / 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。即ち、種々の変異Rep発現ベクターを構築し、AAVS1領域特異的組込み活性を保持し細胞毒性が減弱したものの探索を試みた。具体的には、Rep78のN末側約1/3の領域(部位特異的組込み反応及び細胞毒性に関わるドメインが存在すると考えられる)において、酸性あるいは塩基性のアミノ酸を一つずつアラニンに変換した変異Rep発現ベクターを多数構築した。これらを用いて解析した結果、H18A、RD61AA、R122A、E201A、およびE226Aの各変異Rep発現ベクターを用いた場合に、部位特異的組込み活性が失われずに細胞毒性が減弱する傾向が認められた。現在、さらにこれらの変異体について詳細な解析を進めている。また、TVI法は、分裂細胞に遺伝子導入する場合に大きな価値があると考えられ、造血系細胞で機能するかどうか検討した。即ち、AAV-ITRで挟んだneo^r遺伝子発現ユニットを持つプラスミドとRep78発現プラスミドを共にK562細胞にトランスフェクトし、その後、G418添加培養で得られたクローンについて、neo^r遺伝子のAAVSI領域への特異的組み込みの有無をサザン法およびFISH法にて検討した。その結果、Rep78を発現させた場合にITR-neo^r遺伝子の部位特異的組み込みが観察され、TVI法が造血系細胞でも有効であることが示された。さらに、組込み部位の塩基配列の解析を行っている。

Research Products

(12 results)

All Other

All Publications (12 results)

[Publications] Ogasawara,Y.: "Efficient production of adeno-associated virus vectors using split-type helper plasmids" Jpn.J.Cancer Res.(in press).
[Publications] Matsda,K.M.: "Development of a modified selective amplifier gene for hematopoietic stem cell gene therapy" Gene Ther.(in press).
[Publications] Urabe ,M.: "Charged-to-alanine scanning mutagenesis of N-terminal half of adeno-associated virus type 2 Rep78 protein" J.Virol.(in press).
[Publications] Ogasawara,Y.: "Potential application of dominant negative retinoic acid receptor genes for ex vivo expansion of hematopoietic stem cells" Gene Ther.Mol.Biol.(in press).
[Publications] Ozawa,K.: "Strategies for gene therapy of Parkinson's disease using adeno-associated virus(AAV) vectors" Biogenic Amines. (in press).
[Publications] Fan,D.: "Behavioral recovery in 6-hydroxydopamine-lesioned rats by cotransduction of striatum with tyrosine hydroxylase and aromatic L-amino acid decarboxylase genes using two separate adeno-associated virus vectors" Hum.Gene Ther.9. 2527-2535 (1998)
[Publications] Kodaira,H.: "Fas and mutant estrogen receptor chimeric gene: a novel suicide vector for tamoxifen-inducible apoptosis" Jpn.J.Cancer Res.89. 741-747 (1998)
[Publications] Kokubun,M.: "Apoptosis-mediated regulation of recombinant human granulocyte colony-stimulating factor production by genetically engineered fibroblasts" Gene Ther.5. 923-929 (1998)
[Publications] Ozawa,K.: "A novel selective amplifier gene for hematopoietic stem cell gene therapy" Cancer Res.Ther.Contr.7. 27-31 (1998)
[Publications] Fan,D.: "Prevention of dopaminergic neuron death by adeno-associated virus vector-mediated GDNF gene transfer in rat mesencephalic cells in vitro" Neurosci.Lett.248. 61-64 (1998)
[Publications] Maeda,Y.: "Efficient gene transfer into cardiac myocytes using adeno-associated virus(AAV)vectors" J.Mol.Cell.Cardiol.30. 1341-1348 (1998)
[Publications] Ogasawara,Y.: "The use of heterologous promoters for adeno-associated virus(AAV)protein expression in AAV vector production" Microbiol.Immunol.42. 177-185 (1998)

1998 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] Ogasawara,Y.: "Efficient production of adeno-associated virus vectors using split-type helper plasmids" Jpn.J.Cancer Res.(in press).

[Publications] Matsda,K.M.: "Development of a modified selective amplifier gene for hematopoietic stem cell gene therapy" Gene Ther.(in press).

[Publications] Urabe ,M.: "Charged-to-alanine scanning mutagenesis of N-terminal half of adeno-associated virus type 2 Rep78 protein" J.Virol.(in press).

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

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

[Publications] Fan,D.: "Behavioral recovery in 6-hydroxydopamine-lesioned rats by cotransduction of striatum with tyrosine hydroxylase and aromatic L-amino acid decarboxylase genes using two separate adeno-associated virus vectors" Hum.Gene Ther.9. 2527-2535 (1998)

[Publications] Kodaira,H.: "Fas and mutant estrogen receptor chimeric gene: a novel suicide vector for tamoxifen-inducible apoptosis" Jpn.J.Cancer Res.89. 741-747 (1998)

[Publications] Kokubun,M.: "Apoptosis-mediated regulation of recombinant human granulocyte colony-stimulating factor production by genetically engineered fibroblasts" Gene Ther.5. 923-929 (1998)

[Publications] Ozawa,K.: "A novel selective amplifier gene for hematopoietic stem cell gene therapy" Cancer Res.Ther.Contr.7. 27-31 (1998)

[Publications] Fan,D.: "Prevention of dopaminergic neuron death by adeno-associated virus vector-mediated GDNF gene transfer in rat mesencephalic cells in vitro" Neurosci.Lett.248. 61-64 (1998)

[Publications] Maeda,Y.: "Efficient gene transfer into cardiac myocytes using adeno-associated virus(AAV)vectors" J.Mol.Cell.Cardiol.30. 1341-1348 (1998)

[Publications] Ogasawara,Y.: "The use of heterologous promoters for adeno-associated virus(AAV)protein expression in AAV vector production" Microbiol.Immunol.42. 177-185 (1998)

小澤敬也自治医科大学, 医学部, 教授 (30137707)