1998 年度実績報告書

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

研究課題

研究課題/領域番号	09470520
研究機関	熊本大学
研究代表者	松田一郎熊本大学, 医学部, 名誉教授 (10000986)
研究分担者	島田隆日本医科大学, 教授 (20125074) 遠藤文夫熊本大学, 医学部, 教授 (00176801) 山本哲郎熊本大学, 医学部, 教授 (60112405)
キーワード	遺伝子治療 / ベクター / OTC欠損症 / AAVベクター
研究概要	アデノ随伴ウイルス(以下AAV)はヒトパルボウイルスの一種で約4.7kbの1本鎖DNAのゲノムを持ち、ウイルスの複製の調節に関わっていろり遺伝子と構造蛋白をコードしているcap遺伝子からなる。このウイルスは感染後、ヒト19番染色体S1部位に組み込まれることが分かっている。大きな特色は非分裂細胞にも感染可能で、また感染細胞のゲノムにintegrateされることである。人の先天代謝異常症に対する遺伝子治療にウイルスベクターの使用を考えるとき、アデノウイルスベクターではその効果が一過性であり、またはレトロウルスでは分裂細胞にのみ導入可能なのに対し、AAVでは非分裂性の標的細胞のゲノムに導入され、効果が長期に及ぶことが期待される。そこで、尿素サイクルの酵素の一つであるオルニチントランスカルバミラーゼ(OTC)とチロシン分解酵素のフマリルアセト酢酸水酸化酵素(FAH)の欠損症に対する遺伝子治療にむけてAAVベクターの作製を試みた。組換えAAVの作製にあたり、両端のITRだけを残しrepとcapを取り除き、ヒトOTCとヒトFAHのcDNAをそれぞれ組み込んだvector plasmidを作製した。このvector plasmidを1μgと、repとcapを組み込み、両端のITRを取り除いたhelper plasmidをそれぞれ10μgずつ7×10^6のHeLa細胞にtransfectさせ、G418を用いてselectionし約10^4cfu/mlの組換えAAV/FAHベクターを産生するpackaging cell lineを作製した。現在、さらに感染効率を上げるために、硫酸アンモニウムと塩化セシウムを用いた濃縮法を試みている。高濃度のAAVウイルスを用いてさらに先天代謝異常症の遺伝子治療実験をすすめている。

研究成果
(18件)

すべてその他

すべて文献書誌 (18件)

[文献書誌] Shimizu M.: "Ten novel mutations of the ornithine transcarbamylase(OTC)gene in OTC deficiency." Hum Mutation,Supp. 1. 5-7 (1998)
[文献書誌] Nishiyori A.: "Y55D mutation in ornithine transcarbamylase associated with late-onest hyperammonemia in a male." Hum Mutant Suppl. 1. 131-133 (1998)
[文献書誌] Watanabe A.: "Prenatal diagnosis of ornithine transcarbamylase dificiency by using a single nucleated erythrocyte from maternal blood." Hum Genet. 102. 611-615 (1998)
[文献書誌] Yorifuji T.: "X-inactivation pattern in the liver of a manifesting female with ornithine transcarbamylase(OTC)deficiency." Clin Genet. 54. 349-353 (1998)
[文献書誌] Komaki S.: "The polymorphic 43Thr bcl-2 protein confers relative resistance autoimmnity:an analytical evaluation." Hum Genet. 103. 435-440 (1998)
[文献書誌] Kubo S.: "Hepatocyte injury in tyrosinemia type 1 is induced by fumarylacetate and is inhibited by xaspase inhibitors." Proc Natl Acad Sci. 95. 9552-9557 (1998)
[文献書誌] Nagano K.: "Intracellular distribution of Wilson's disease gene product(ATPase7B)after in vitro and in vivo exogenous expression in hepatocytes from the LEC rat,an animal model of Wilson's disease." Hepatology. 27. 799-807 (1998)
[文献書誌] Uchino T.: "Neurodevelopmental outcome of long-term therapy of urea cycle disorders in Japan." J Inherit metabol disease. 21. 151-159 (1998)
[文献書誌] Goseki-Sone M.: "Hypophosphatasia:identification of five novel missense mutations in the tissue-nonspecific alkaline phosphatase gene among Japanese patients.Hum." Mutat.Suppl. 1. 263-267 (1998)
[文献書誌] Tanosaki,S.: "Relationship of microsatellite instebility to N-ras mutation and duration of disease-free survivial in acute leukemia." Cancer. 83. 475-481 (1998)
[文献書誌] Miyake,K.: "Stable integration of HIV-retrovial vectors into the chromosomes of non-dividing cells." Hum.Gene.Ther.9. 467-475 (1998)
[文献書誌] Matsuoka,H.: "An improved methods of HIV vector mediated gene trasnfer." Inter.J.Hematol.67. 267-273 (1998)
[文献書誌] Orimo,H.: "A novel missense mutation and frameshift mutations in the type II receptor of transforming growth factor-b gene in sporadic colon cancer with microsatellite instability." Mutat.Res.Genomics. 382. 115-120 (1998)
[文献書誌] Goseki-Sone,M.: "Expression of the mutant(1735T-del)tissue-nonspecific alkaline phosphatase gene from hypophosphatasia patients." J.Bone Miner.Res.13. 1827-1834 (1998)
[文献書誌] Ikeda,M.: "Close correlation between mutations of E2F4 and hMSH3 genes in colorectal cancers with microsatellite instability." Cancer Res.58. 594-598 (1998)
[文献書誌] Shimizu,T.: "A simple and efficient purification of transduced cells by using green fluorescent protein(GFP)gene as a selectable marker." Acta Paediatr.Jpn.40. 586-592 (1998)
[文献書誌] Igarashi,T.: "A novel strategy of cell targeting based on tissue specific expression of the ecotropic retrovirus receptor gene." Human Gene Ther.9. 2691-2698 (1998)
[文献書誌] Koseki,S.: "Factors governing the activity in vivo of ribozymes transcribed by RNA polymeraseIII." J.Viol.73. 1868-1877 (1998)

1998 年度 実績報告書

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

研究代表者

松田 一郎 熊本大学, 医学部, 名誉教授 (10000986)

研究成果

[文献書誌] Shimizu M.: "Ten novel mutations of the ornithine transcarbamylase(OTC)gene in OTC deficiency." Hum Mutation,Supp. 1. 5-7 (1998)

[文献書誌] Nishiyori A.: "Y55D mutation in ornithine transcarbamylase associated with late-onest hyperammonemia in a male." Hum Mutant Suppl. 1. 131-133 (1998)

[文献書誌] Watanabe A.: "Prenatal diagnosis of ornithine transcarbamylase dificiency by using a single nucleated erythrocyte from maternal blood." Hum Genet. 102. 611-615 (1998)

[文献書誌] Yorifuji T.: "X-inactivation pattern in the liver of a manifesting female with ornithine transcarbamylase(OTC)deficiency." Clin Genet. 54. 349-353 (1998)

[文献書誌] Komaki S.: "The polymorphic 43Thr bcl-2 protein confers relative resistance autoimmnity:an analytical evaluation." Hum Genet. 103. 435-440 (1998)

[文献書誌] Kubo S.: "Hepatocyte injury in tyrosinemia type 1 is induced by fumarylacetate and is inhibited by xaspase inhibitors." Proc Natl Acad Sci. 95. 9552-9557 (1998)

[文献書誌] Nagano K.: "Intracellular distribution of Wilson's disease gene product(ATPase7B)after in vitro and in vivo exogenous expression in hepatocytes from the LEC rat,an animal model of Wilson's disease." Hepatology. 27. 799-807 (1998)

[文献書誌] Uchino T.: "Neurodevelopmental outcome of long-term therapy of urea cycle disorders in Japan." J Inherit metabol disease. 21. 151-159 (1998)

[文献書誌] Goseki-Sone M.: "Hypophosphatasia:identification of five novel missense mutations in the tissue-nonspecific alkaline phosphatase gene among Japanese patients.Hum." Mutat.Suppl. 1. 263-267 (1998)

[文献書誌] Tanosaki,S.: "Relationship of microsatellite instebility to N-ras mutation and duration of disease-free survivial in acute leukemia." Cancer. 83. 475-481 (1998)

[文献書誌] Miyake,K.: "Stable integration of HIV-retrovial vectors into the chromosomes of non-dividing cells." Hum.Gene.Ther.9. 467-475 (1998)

[文献書誌] Matsuoka,H.: "An improved methods of HIV vector mediated gene trasnfer." Inter.J.Hematol.67. 267-273 (1998)

[文献書誌] Orimo,H.: "A novel missense mutation and frameshift mutations in the type II receptor of transforming growth factor-b gene in sporadic colon cancer with microsatellite instability." Mutat.Res.Genomics. 382. 115-120 (1998)

[文献書誌] Goseki-Sone,M.: "Expression of the mutant(1735T-del)tissue-nonspecific alkaline phosphatase gene from hypophosphatasia patients." J.Bone Miner.Res.13. 1827-1834 (1998)

[文献書誌] Ikeda,M.: "Close correlation between mutations of E2F4 and hMSH3 genes in colorectal cancers with microsatellite instability." Cancer Res.58. 594-598 (1998)

[文献書誌] Shimizu,T.: "A simple and efficient purification of transduced cells by using green fluorescent protein(GFP)gene as a selectable marker." Acta Paediatr.Jpn.40. 586-592 (1998)

[文献書誌] Igarashi,T.: "A novel strategy of cell targeting based on tissue specific expression of the ecotropic retrovirus receptor gene." Human Gene Ther.9. 2691-2698 (1998)

[文献書誌] Koseki,S.: "Factors governing the activity in vivo of ribozymes transcribed by RNA polymeraseIII." J.Viol.73. 1868-1877 (1998)

1998 年度実績報告書

松田一郎熊本大学, 医学部, 名誉教授 (10000986)