| Research Abstract |
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
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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
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