Retrovirus-Mediated Gene Transfer of 6-Pyruvoyl-Tetrahydropterin Synthase Corrects Tetrahydrobiopterin Deficiency in Fibroblasts from Hyperphenylalaninemic Patients

Tetrahydrobiopterin (BH₄) deficiency, a variant form of hyperphenylalaninemia with progressive neurological dysfunction, is primarily caused by autosomal recessive mutations in the gene encoding the 6-pyruvoyl-tetrahydropterin synthase (PTPS). PTPS is a biosynthetic enzyme for the BH₄ co-factor, and its deficiency is associated with a malfunction of the phenylalanine catabolism in the liver and a lack of biogenic amine neurotransmitters dopamine and serotonin in the brain. We have previously isolated the wild-type PTPS cDNA and identified several mutations responsible for a decreased enzyme activity in patients. This study reports the in vitro correction of BH₄ deficiency by using retrovirus mediated transfer of the PTPS cDNA into primary fibroblast cultures established from different patients. The Bing packaging cell line was used for amphotropic virus production. Following PTPS gene transfer, stimulation with cytokines restored biosynthesis of BH₄ in originally defective cells to values comparable to those of heterozygous fibroblasts from clinically healthy subjects. These results not only provide a direct proof that the mutations in PTPS were causative for the mutant phenotype, but they are also the first step toward gene therapy as a potential alternative approach to treat BH₄ deficiency.

Tetrahydrobiopterin (BH₄) is required as a cofactor for a number of enzymes, like the aromatic amino acid hydroxylases and the nitric oxide synthases, effective in neuronal and peripheral organs. BH₄ deficiency is a rare severe genetic disorder leading primarily to hyperphenylalaninemia that is nontreatable with dietary restrictions of phenylalanine. Because synthetic BH₄ replacement therapy is complex and may only be partially effective, development of new treatment strategies is required. We have not only studied BH₄ deficiency in humans but also investigated genetically and biochemically its most common defective biosynthetic enzyme, the 6-pyruvoyl-tetrahydropterin synthase (PTPS), as a target for retrovirus-mediated gene transfer in vitro. Following efficient and successful gene delivery into primary patients' fibroblasts, BH₄ production was observed, thus demonstrating that the biosynthetic pathway was restored. These results indicate the potential usefulness of somatic gene therapy for this disease.