GENE THERAPY ON HEPATIC ENZYME DEFICIENCY.

我们利用PKU模型小鼠研究了肝酶缺乏症的基因治疗。我们用粘附盒方法构建了在CAG启动子控制下携带人PAH基因的复制缺陷重组腺病毒。重组病毒在体外感染COS7细胞产生相当于正常肝细胞的PAH活性。将含1.2×10~(-9)p.f.u的病毒溶液注入PKU模型小鼠尾静脉时，主要在肝脏中检测到PAH基因和酶活性。血清苯丙氨酸浓度在24小时内降至正常。然而，生化变化只持续了10天，由于宿主对腺病毒的免疫反应，再次注射病毒未能纠正高苯丙氨酸血症。小鼠每日给予免疫抑制剂FK506后，基因表达持续时间延长至35d以上，且重复基因传递可降低血清苯丙氨酸水平。在这些实验中，色素减少的PKU小鼠表现出明显的被毛色素沉着，从灰色到黑色，与FK506处理无关。表型逆转可能是由于酪氨酸代谢的改善。色素减退的持续时间与苯丙氨酸恢复正常的时间密切相关。本研究首次证实了通过基因转移收集PKU小鼠的生理表型，提示了基因治疗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.