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，临床表现为深度智力低下和皮肤和头发色素沉着。作为PKU基因治疗的一步，我们构建了一种复制缺陷的重组腺病毒，在CAG启动子（Adex1CA-Y-hPAH）的控制下，含有人PAH cDNA。将重组腺病毒注射到PKU模型小鼠（Pah^<enu2>）的尾静脉中，成功地恢复了Pah在肝脏中的活性，使血清苯丙氨酸水平正常化，随后纠正了毛色的低色素沉着。然而，通过western blot分析和血清转氨酶测定，它也引起了宿主对重组病毒和人多环芳烃的深刻免疫反应。给小鼠注射免疫抑制剂FK506，成功阻断了宿主的免疫反应，显著延长了基因表达的持续时间，并允许基因重复传递。我们进一步研究了多环芳烃的辅助因子四氢生物蝶呤（BH_4）对PKU小鼠基因治疗的影响。我们首先通过尾静脉给药给BH_4给药，使小鼠获得Adex1CA-Y-hPAH。仅1 × 10^8 pfu /小鼠的病毒就能降低血清苯丙氨酸水平。然而，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对治疗效果的增强可能是由于改善了转导肝细胞中BH_4的相对缺乏，并激活了内在BH_4浓度明显低的肝外组织中的PAH。然后，我们对添加或不添加BH_4的PKU小鼠肌肉注射AdexlCA-Y-hPAH。血清苯丙氨酸水平仅在同时给药BH_4时才降低。目前的研究表明，PKU可能通过“异位”基因表达来治疗，允许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