Gene analysis and gene therapy in tyrosinemias.

我们分别从人、小鼠和猪肝脏的mRNA文库中克隆了人、小鼠和猪的4-羟基苯丙酮酸双加氧酶的基因文库，并以人和小鼠的基因文库为基础，获得了含有HpD外显子的DNA片段，从而阐明了人和小鼠HpD基因的结构。基于这些研究进展，我们分析了一例日本1型酪氨酸血症患者的人FAH基因突变和一只酪氨酸血症III小鼠的小鼠HpD基因突变。这些研究分别发现了FAH基因和HpD基因的突变。使用Fah缺陷的非致死小鼠和HpD缺陷的III小鼠，我们产生了同时携带FAH基因和Hpd基因突变的小鼠。FAH基因缺陷小鼠对新生小鼠具有致死性，但这两个突变体是有效的，说明hpd基因缺陷挽救了白化缺失小鼠的致死表型.进一步，我们构建了表达人hpd的重组腺病毒.在培养细胞中转导有效表达人hpd，该重组病毒可用于在实验动物中表达人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.