Adenovirus-mediated gene transfer in phenylketonuria model mice

苯丙酮尿症（PKU）是由肝脏苯丙氨酸羟化酶（PAH）缺乏引起的，临床上以严重的智力低下和皮肤毛发色素减退为特征。作为PKU基因治疗的一步，我们构建了一个复制缺陷型重组腺病毒，携带人PAH cDNA的有效CAG启动子控制下（Adex 1CA-Y-hPAH）。将重组腺病毒尾静脉注射到PKU模型小鼠（Pah<enu2>）中，成功地恢复了肝脏中PAH的活性，使血清苯丙氨酸水平正常化，随后纠正了毛色减退。然而，它也引起了深刻的宿主对重组病毒和人PAH的免疫应答，如通过蛋白质印迹分析和血清转氨酶的测量所确定的。给小鼠施用免疫抑制剂FK 506成功地阻断了宿主免疫应答，显著延长了基因表达的持续时间，并允许重复基因递送。我们 ...更多信息 然后观察PAH辅因子四氢生物蝶呤（tetrahydrobiopterin，BH_4）对PKU小鼠基因治疗的影响。我们首先将BH_4通过尾静脉口服给药于接受Adex 1CA-Y-hPAH的小鼠。低至1x10^8 p.f.u./小鼠血清苯丙氨酸水平明显降低。而在不添加BH_4的情况下，相同量的重组病毒不能改变苯丙氨酸的浓度。补充BH_4可改善BH_4在肝细胞内的相对缺乏，并激活内源性BH_4浓度较低的肝外组织中的PAH，从而增强BH_4的治疗效果。然后，我们对补充或不补充BH_4的PKU小鼠进行AdexlCA-Y-hPAH肌肉注射。血清苯丙氨酸水平仅在同时给予BH_4时才有所降低。目前的研究表明，PKU可以通过“异位”基因表达来治疗，从而使基因治疗更容易进入器官而不是肝脏。少

Phenylketonuria (PKU) is caused by deficiency of hepatic phenylalanine hydroxylase (PAH) and clinically characterized by profound mental retardation and hypopigmentation of skin and hair. As a step toward gene therapy for PKU, we constructed a replication-defective, recombinant adenovirus harboring human PAH cDNA under the control of a potent CAG promoter (Adex1CA-Y-hPAH). Injection of the recombinant adenovirus into tail veins of PKU model mice (Pah^<enu2>) successfully restored the PAH activity in liver, normalized the serum phenylalanine level and, subsequently, corrected the hypopigmentation of coat color. However, it also provoked a profound host immune response against the recombinant virus and human PAH, as determined by western blot analysis and the measurement of serum transaminase. Administration of an immunosuppressant, FK506, to mice successfully blocked the host immune response, significantly prolonged the duration of gene expression and allowed repeated gene delivery. We … More then examined the effect of tetrahydrobiopterin (BH_4), a cofactor for PAH, on gene therapy of PKU mice. We first administered BH_4 orally into mice which received Adex1CA-Y-hPAH through the tail vein. As little as 1x10^8 p.f.u./mouse of the virus were able to decrease the serum phenylalanine level. Without BH_4 supplementation, however, the same amount of the recombinant virus failed to alter phenylalanine concentration. The enhancement of therapeutic effect with BH_4 supplementation might be explained by the amelioration of relative BH_4 deficiency in transduced hepatocytes and the activation of PAH in extrahepatic tissues where intrinsic BH_4concentration is significantly low. We then performed intramuscular injection of AdexlCA-Y-hPAH on PKU mice supplemented with or without BH_4. Reduction of serum phenylalanine level was observed only when BH_4 was co-administered. The current study suggested that PKU may be treated by "ectopic" gene expression, allowing gene therapy in more readily accessible organs rather than liver. Less