Reconstitution of a metabolic pathway with triple-cistronic IRES-containing retroviral vectors for correction of tetrahydrobiopterin deficiency.

BACKGROUND: Tetrahydrobiopterin (BH4) is an essential cofactor for catecholamine and serotonin neurotransmitter biosynthesis. BH4 biosynthesis is carried out in a three-enzyme pathway involving GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SR). Treatment of genetic defects leading to BH4 deficiency requires neurotransmitter replacement since synthetic cofactor does not efficiently penetrate the blood-brain barrier. Autologous fibroblasts transplanted into the brain as depository cells for drug delivery might offer an alternative. However, normal fibroblasts do not express GTPCH, and fibroblasts from PTPS patients lack two biosynthetic enzymes for BH4 production.

METHODS: We engineered primary fibroblasts by the use of triple-cistronic, retroviral vectors for cofactor production.

RESULTS: Constitutive SR activity in these cells enabled BH4 biosynthesis by transducing GTPCH and PTPS cDNAs together with a selective marker coupled in a single transcript with two IRES-elements in tandem. Upon reaching a critical concentration (> 400 pmol/mg protein) of intracellular BH4, the fibroblasts efficiently released cofactor even under non-dividing conditions.

CONCLUSION: The use of triple-cistronic vectors for single transduction to reconstitute metabolic pathways or to treat multi-genetic diseases may be useful for engineering, for instance, depository cells for various organs, including the nervous system.