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- Title
Development of a skin-based metabolic sink for phenylalanine by overexpression of phenylalanine hydroxylase and GTP cyclohydrolase in primary human keratinocytes.
- Authors
Christensen, R; Kolvraa, S; Blaese, R M; Jensen, T G
- Abstract
Phenylketonuria, PKU, is caused by deficiency of phenylalanine hydroxylase (PAH) resulting in increased levels of phenylalanine in body fluids. PAH requires the non-protein cofactor BH[sub 4] and the rate-limiting step in the synthesis of BH[sub 4] is GTP cyclohydrolase I (GTP-CH). Here we show that overexpression of the two enzymes PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH[sub 4] supplementation. Integration of multiple PAH and GTP-CH transgenes were achieved after optimized retroviral transduction. Phenylalanine clearance was measured ex vivo in primary human keratinocytes cotransduced with PAH and GTP-CH (more than 370 nmol/24 h/10[sup 6] cells), a level exceeding that of a human fiver cell line (HepG2 cells). Cells overexpressing either one of the enzymes alone did not clear significant amounts of phenylalanine. Transfer of the two genes into the same cell was not necessary, since cocultivation of cells transduced separately with PAH and GTP-CH also resulted in phenylalanine clearance. Thus the experiments indicate metabolic cooperation between cells overexpressing PAH and cells overexpressing GTP-CH, possibly due to intercellular transport of synthesized BH[sup 4].
- Subjects
ENZYMES; KERATINOCYTES; PHENYLKETONURIA
- Publication
Gene Therapy, 2000, Vol 7, Issue 23, p1971
- ISSN
0969-7128
- Publication type
Article
- DOI
10.1038/sj.gt.3301337