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Reconstitution of a metabolic pathway with triple‐cistronic IRES‐containing retroviral vectors for correction of tetrahydrobiopterin deficiency
Author(s) -
Laufs Stephanie,
Kim Seon Hee,
Kim Sunyoung,
Blau Nenad,
Thöny Beat
Publication year - 2000
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/(sici)1521-2254(200001/02)2:1<22::aid-jgm86>3.0.co;2-1
Subject(s) - tetrahydrobiopterin , gtp cyclohydrolase i , biopterin , cofactor , biology , microbiology and biotechnology , biosynthesis , proenkephalin , enzyme , biochemistry , chemistry , enkephalin , receptor , opioid
Background Tetrahydrobiopterin (BH 4 ) is an essential cofactor for catecholamine and serotonin neurotransmitter biosynthesis. BH 4 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 BH 4 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 BH 4 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 BH 4 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 BH 4 , 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. Copyright © 2000 John Wiley & Sons, Ltd.