GENE THERAPY ON HEPATIC ENZYME DEFICIENCY.

肝酶缺乏症的基因治疗。

基本信息

批准号：
08457218
负责人：
NARISAWA Kuniaki
金额：
$ 4.93万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457218/
关键词：
HEPATIC ENZYME DEFICIENCY PHENYLKETONURIA GENE THERAPY ADNOVIRUS VECTOR SERUM PHENYLALANINE COAT COLOR

项目摘要

We investigated gene therapy on hepatic enzyme deficiency using PKU model mice. We constructed a replication-defective recombinant adenovirus harboring human PAH cDNA under the control of a potent CAG promoter using a cosmid-cassette method. Infection of COS7 cells with the recombinant virus at m.o.i=3.0 in vitro produced the PAH activity equivalent to normal hepatocytes. When the solution containing 1.2x10^9 p.f.u of the virus was infused into the tail vein of PKU model mice, PAH cDNA and enzymatic PAH activity were mainly detected in liver. Serum phenylalanine concentration decreased to normal level within 24 hrs. However, the biochemical change lasted for only 10 days and re-administration of the virus failed to correct hyperphenylalaninemia due to host immune response against the adenovirus. When the mice were treated with daily administration of an immunosuppressant FK506, the duration of gene expression was prolonged to more than 35 days and repeated gene delivery was able to decrease the serum phenylalanine level. In these experiments, hypopigmented PKU mice showed distinct pigmentation of coat, from grayish color to black, regardless of FK506 treatment. The phenotypic reversal was presumably due to improved tyrosine metaboLism. The duration of hypopigmentation closely correlated with that of the normalization of phenylalanine level. Our study is the first to demonstrate the collection of physical phenotype in PKU mice by gene transfer, suggesting the feasibility of gene therapy on PKU.

我们使用PKU模型小鼠研究了基因治疗在肝酶缺乏症上。我们使用cosmid-costette方法构建了一种在有效的CAG启动子的控制下构建了具有人类PAH cDNA的复制缺陷型腺病毒。在M.O.I = 3.0体外，用重组病毒感染COS7细胞的PAH活性等于正常的肝细胞。当将含有1.2x10^9 p.f.u的溶液注入PKU模型小鼠的尾静脉中时，PAH cDNA和酶促PAH活性主要在肝脏中检测到。血清苯丙氨酸浓度在24小时内降至正常水平。然而，生化变化仅持续了10天，由于宿主对腺病毒的免疫反应，该病毒的重新给药未能纠正过度苯基丙氨酸。当小鼠每天用免疫抑制剂FK506治疗小鼠时，基因表达的持续时间延长至35天以上，并且反复的基因递送能够降低血清苯丙氨酸水平。在这些实验中，不适式的PKU小鼠表现出从灰色到黑色的外套的独特色素沉着，无论FK506处理如何。表型逆转大概是由于改善酪氨酸代谢。异形的持续时间与苯丙氨酸水平的标准化密切相关。我们的研究是第一个通过基因转移来证明PKU小鼠中物理表型的一项研究，这表明基因治疗对PKU的可行性。