Fig. 4.

Phenotype correction of severe hyperphenylalaninemia in PKU mice after treatment with an integrating plasmid expressing mouse PAH cDNA. (A) Plasmid map of pCmPAH-B. The vector contains a mouse PAH gene cDNA expression cassette driven by the CAG promoter (31) and a wild-type attB sequence. (B) Serum phenylalanine curves of treated PKU mice. Each mouse received injections of either PBS or pCmPAH-B at three different time points (shown with arrow). Serum phenylalanine levels decreased abruptly in all groups (n = 6 per group) after each PAH vector administration. In the integrase-negative treatment group, serum phenylalanine concentrations returned to pretreatment levels (P > 0.1) within 2 weeks. In the integrase-positive treatment group, serum phenylalanine concentrations remained stable at a substantially reduced level (P < 0.01). After three administrations of the vector, serum phenylalanine levels in the integrase-positive treatment group decreased to the normal range (P > 0.1 when compared with that of normal mice) and remained at those levels thereafter. (C) Fur color change in PKU mice after treatment. After three consecutive administrations of the integrating plasmid DNA vector, no mice changed fur color in the integrase-negative control treatment group (Upper), whereas all PKU mice in the vector-treatment group (Lower) turned black. (D) PAH activities in liver extracts of normal and treated PKU mice. Specific enzymatic activities were defined as total cpm in [¹⁴C]tyrosine produced in the assay per mg of protein in the liver extracts, and the initial slopes of the saturation curves were compared statistically by the Student t test (P < 0.01). The cpm/mg protein values were calculated from the tangents of the curves. Each groups contained six animals. In treated PKU mice the total enzymatic activity was ≈17% of normal, whereas in the liver of heterozygous mice the level was 50% of normal as expected. In untreated PKU mice, the level was <1% of normal.