Gene analysis and gene therapy in tyrosinemias.

酪氨酸血症的基因分析和基因治疗。

• 批准号: 05671886
• 负责人: ENDO Fumio
• 金额: $ 1.28万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05671886/
• 关键词: tyrosinemia; gene mutation; gene structure; animal model; recombinant adenovirus; gene therapy; チロジン; 遺伝子; 突然変異; モデルマウス; HPD

We have clone cDNA for human, mouse and porcine 4-hydroxyphenylpyruvate dioxygenase from cDNA libralies constructed from mRNA of human, mouse and porcine liver respectively, Using human and mouse cDNA as proves, we obstained DNA fragments containing exons of HPD from human and mouse genomic libraries, Through these studies we have clarified the structure of human and mouse HPD gene. Similarly, we have elucidated the structures of cDNA and gene for human fumarylacetoacetase.Based on these progress we analyzed gene mutations in human FAH gene from a Japanese patient with type 1 tyrosinemia and mouse HPD gene from III mouse, a tyrosinemic mouse.These studies revealed mutations on FAH gene and HPD gene, respectively.Using abino lethal mouse which is defective for Fah, and III mouse which is defective for HPD,we generated mice which carry both mutation, one on FAH gene and one on HPD gene. Fah deficient mice were neonatally lethal but these double mutants were vaiable, indicating that defective HPD gene rescues the lethal phenotype of albino deletion mouse.Futher, we constructed recombinant adenovirus which express human HPD.In cultured cells, transduction of the recombinant virus effectively expresses human HPD.This recombinant virus will be useful to express human HPD in experimental animals.

我们分别从人、小鼠和猪肝脏mRNA构建的cDNA文库中克隆了人、小鼠和猪4-羟基苯丙酮酸双加氧酶的cDNA，以人和小鼠的cDNA为证明，从人和小鼠基因组文库中获得了含有HPD外显子的DNA片段，通过这些研究，我们阐明了人和小鼠HPD基因的结构。在此基础上，我们对日本1型酪氨酸血症患者的FAH基因和酪氨酸血症小鼠III型小鼠的HPD基因进行了基因突变分析，分别发现了FAH基因和HPD基因的突变。我们产生了携带两种突变的小鼠，一种在FAH基因上，一种在HPD基因上。Fah基因缺陷小鼠具有致死性，而这些双突变体却具有致死性，说明HPD基因缺陷挽救了白化病缺失小鼠的致死性表型。进一步构建了表达人HPD的重组腺病毒，在培养的细胞中转导，重组病毒可有效地表达人HPD，为在实验动物中表达人HPD奠定了基础。

项目成果

Endo F.,et al.: "Exess copper and ceruloplasmin biosynthesis in long-term cultured hepatocytes from Long-Evans Cinnamon(LEC)rats,a model of Wilson disease." J.Biol.Chem.(in press).

Endo F. 等人：“长期培养的 Long-Evans Cinnamon (LEC) 大鼠（威尔逊病模型）的肝细胞中存在过量的铜和铜蓝蛋白生物合成。”

Endo F., et al.: "Structure of he human 4-hydroxyphenylpyruvic acid dioxygenase (HPD)" Genomics. 23. 534-539 (1994)

Endo F. 等人：“人 4-羟基苯基丙酮酸双加氧酶 (HPD) 的结构”基因组学。

Endo F., et al.: "Structural organization and analysis of human fumarylacetoacetate hydrolase gene in tyrosinemia type 1." Biochem.Biophys.Acta.1226. 168-172 (1994)

Endo F. 等人：“1 型酪氨酸血症中人延胡索酰乙酰乙酸水解酶基因的结构组织和分析”。

Endo F.,et al.: "Characterization of a point mutation in the pyruvate El α gene from two boys with primary lactic acidemia." J.Inher.Metab.Dis.17. 189-195 (1994)

Endo F. 等人：“两个患有原发性乳酸血症的男孩的丙酮酸 El α 基因点突变的特征。”J.Inher.Metab.Dis.189-195 (1994)。

Endo F., et al.: "Measurement of blood holoceruloplasmin by E_1 alpha using a mouse monoclonal antibody directed to holoceruloplasmin. Implication for mass screening of Wilson disease." J.Inher.Metab.Dis.17. 616-620 (1994)

Endo F. 等人：“使用针对全铜蓝蛋白的小鼠单克隆抗体通过 E_1 α 测量血液全铜蓝蛋白。对威尔逊病大规模筛查的意义。”