Development of a skin-based metabolic sink for phenylalanine by overexpression of phenylalanine hydroxylase and GTP cyclohydrolase in primary human keratinocytes

Phenylketonuria, PKU, is caused by deficiency of phenylalanine hydroxylase (PAH) resulting in increased levels of phenylalanine in body fluids. PAH re quires the non-protein cofactor BH4 and the rate-limiting step in the synth esis of BH4 is GTP cyclohydrolase I (GTP-CH). Here we show that overexpress ion of the two enzymes PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH4 supplementation. Inte gration of multiple PAH and GTP-CH transgenes were achieved after optimized retroviral transduction. Phenylalanine clearance was measured ex vivo in p rimary human keratinocytes cotransduced with PAH and GTP-CH (more than 370 nmol/24 h/10(6) cells), a level exceeding that of a human liver 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 se parately with PAH and GTP-GH also resulted in phenylalanine clearance. Thus the experiments indicate metabolic cooperation between cells overexpressin g PAH and cells overexpressing GTP-CH possibly due to intercellular transpo rt of synthesized BH4.