GENE THERAPY ON HEPATIC ENZYME DEFICIENCY.

我们用PKU模型小鼠研究基因治疗肝酶缺乏症的方法。我们在强效CAG启动子的控制下，利用cosmid-cassette方法构建了一种携带人PAH cDNA的复制缺陷重组腺病毒。重组病毒在m.o i=3.0条件下感染COS7细胞，其PAH活性与正常肝细胞相当。将含有1.2x10^ 9pfu病毒的溶液注入PKU模型小鼠的尾静脉，主要检测肝脏中PAH cDNA和酶促PAH活性。血清苯丙氨酸浓度在24小时内降至正常水平。然而，这种生化变化只持续了10天，由于宿主对腺病毒的免疫反应，再次给药未能纠正高苯丙氨酸血症。当小鼠每天服用免疫抑制剂FK506时，基因表达的持续时间延长至35天以上，重复的基因传递能够降低血清苯丙氨酸水平。在这些实验中，无论FK506治疗与否，低色素PKU小鼠的皮毛都呈现出明显的色素沉着，从灰色到黑色。表型逆转可能是由于酪氨酸代谢的改善。低色素沉着的持续时间与苯丙氨酸水平正常化的持续时间密切相关。我们的研究首次证明了通过基因转移收集PKU小鼠的物理表型，提示基因治疗PKU的可行性。

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.