1997 Fiscal Year Annual Research Report

アデノ随伴ウイルス(AAV)ベクターを用いた尿素サイクル異常症の遺伝子治療研究

Research Project

Project/Area Number	09470520
Research Institution	Kumamoto University
Principal Investigator	松田一郎熊本大学, 医学部, 教授 (10000986)
Co-Investigator(Kenkyū-buntansha)	島田隆日本医科大学, 教授 (20125074) 遠藤文夫熊本大学, 医学部付属病院, 講師 (00176801) 山本哲郎熊本大学, 医学部, 教授 (60112405)
Keywords	遺伝子治療 / ベクター / OTC欠損症 / AAVベクター
Research Abstract	ヒトOTC遺伝子を持つAAVのvector plasmidとしてITR-ITR間が5.35kbでTK-neo遺伝子を持つもの(以下OTC2)と、4.4kbでTK-neoを持たないもの(以下OTC3)の2つを作製した。これらのconstructionをscreeningする目的でリン酸カルシウム法を用いてそれぞれのベクターをpackaging plasmidとともにHeLa細胞にco-transfectionし、野性株アデノウイルスを感染させて、AAVを作製した。OTC2を用いて作製したAAV(以下AAV/OTC2)は3T3細胞を用いて力価測定したところ、10^4cfu/mlであった。一方、OTC3を用いて作製したAAV(以下AAV/OTC3)をdot blot法によりperticle数を測定したところ10^5perticles/mlであった。AAV/OTC2及びAAV/OTC3をそれぞれを2mlずつ10^6個のHepG2細胞にtrunsductionしOTC活性を測定したところ、前者では測定感度以下であったが、後者では1.265μmol/mg protein/hrの活性が得られた。そこで、AAV/OTC3を150ml作製し、硫酸セルロースカラムを用いて濃縮したものを20μl用い、10^6個のHepG2細胞にtransductionしたが0.144μmol/mg protein/hrの活性しか得られなかった。現在、OTC2とpackaging plasmidのpackaging cell lineを作製中であり、より高い力価のAAV/OTCを作製を目指している。

Research Products
(9 results)

All Other

All Publications (9 results)

[Publications] Kubo S.: "In vivo correction of 4-hydroxyphenylpyruvic acid dioxygenase deficiencies in III mice with recombinant adenovirus." Hum. Gene Therapy. 8. 65-71 (1997)
[Publications] Komaki, S.: "Familial lethal inheritance of a mutated paternal gene in females causing X-linked ornithine transcarbamylase(OTC)gene." Am. J. Med. Genet.69. 177-181 (1997)
[Publications] Nanji, M. S.: "Haplotype and mutation analysis in Japanese patients with Wilson Disease." Am. J. Hum. Genet.60. 1423-1429 (1997)
[Publications] Nagano, K.: "Gene mediated expression and partial characterization of ATPase 7B in cultured cells." The Journal of Trace Elements in Experimental Medicine. 10. 111-117 (1997)
[Publications] Endo, F.: "Complete rescue of lethal albino c14CoS mice by null mutation of 4-hydroxyphenypyruvate dioxigenase gene and induction of apoptosis of hepatocytes in these mice by in vivo retrieval of tyrosine catabolic pathway." J. Biol. Chem.272. 24426-24432 (1997)
[Publications] Abe T.: "Transduction of retrovirus-mediated NeoR gene into CD34+ cells purified from granulocyte colony-stimulating factor(G-CSF)-mobilized infant and cord blood." Experimental Hematology. 25. 966-971 (1997)
[Publications] Yamamoto S.: "Herpes simplex birus thymidine kinase/ganciclovir-mediated killing of tumor cell induces tumor-specific cytotoxic T cells in mice." Cancer Gene Therapy.4. 91-96 (1997)
[Publications] Miyake K.: "Stable integration of HIV-based retrovital vectors into the chromasomes of non-dividing cells." Human Gene Therapy. (in press). (1998)
[Publications] Matsuoka K.: "An improved methods of HIV vector mediated gene trasfer." Inter J. Hematol.(in press). (1998)

1997 Fiscal Year Annual Research Report

アデノ随伴ウイルス(AAV)ベクターを用いた尿素サイクル異常症の遺伝子治療研究

Principal Investigator

松田 一郎 熊本大学, 医学部, 教授 (10000986)

Research Products

[Publications] Kubo S.: "In vivo correction of 4-hydroxyphenylpyruvic acid dioxygenase deficiencies in III mice with recombinant adenovirus." Hum. Gene Therapy. 8. 65-71 (1997)

[Publications] Komaki, S.: "Familial lethal inheritance of a mutated paternal gene in females causing X-linked ornithine transcarbamylase(OTC)gene." Am. J. Med. Genet.69. 177-181 (1997)

[Publications] Nanji, M. S.: "Haplotype and mutation analysis in Japanese patients with Wilson Disease." Am. J. Hum. Genet.60. 1423-1429 (1997)

[Publications] Nagano, K.: "Gene mediated expression and partial characterization of ATPase 7B in cultured cells." The Journal of Trace Elements in Experimental Medicine. 10. 111-117 (1997)

[Publications] Endo, F.: "Complete rescue of lethal albino c14CoS mice by null mutation of 4-hydroxyphenypyruvate dioxigenase gene and induction of apoptosis of hepatocytes in these mice by in vivo retrieval of tyrosine catabolic pathway." J. Biol. Chem.272. 24426-24432 (1997)

[Publications] Abe T.: "Transduction of retrovirus-mediated NeoR gene into CD34+ cells purified from granulocyte colony-stimulating factor(G-CSF)-mobilized infant and cord blood." Experimental Hematology. 25. 966-971 (1997)

[Publications] Yamamoto S.: "Herpes simplex birus thymidine kinase/ganciclovir-mediated killing of tumor cell induces tumor-specific cytotoxic T cells in mice." Cancer Gene Therapy.4. 91-96 (1997)

[Publications] Miyake K.: "Stable integration of HIV-based retrovital vectors into the chromasomes of non-dividing cells." Human Gene Therapy. (in press). (1998)

[Publications] Matsuoka K.: "An improved methods of HIV vector mediated gene trasfer." Inter J. Hematol.(in press). (1998)

松田一郎熊本大学, 医学部, 教授 (10000986)