Manipulation of the phenylalanine metabolism in human keratinocytes by retroviral mediated gene transfer.
Phenylketonuria (PKU) is an inherited disease causing increased levels of phenylalanine in body fluids due to deficiency of hepatic phenylalanine hydroxylase (PAH) or other enzymes involved in the phenylalanine metabolism. With the long-term goal of using gene transfer to the skin to remove phenylalanine, we have previously shown that overexpression of PAH, catalyzing the hydroxylation of phenylalanine, and GTP cyclohydrolase (GTP-CH), involved in the formation of the necessary cofactor BH4,are required. Here we investigate whether manipulation of additional steps in the phenylalanine clearance pathway can further improve the phenylalanine uptake and metabolism. Transport of phenylalanine into human keratinocytes could be increased by overexpressing the two subunits LAT1 and 4F2hc of the large neutral amino acid transporter. The PAH enzyme activity was titrated by employing mutant PAH enzymes with different specific activity and by increasing the PAH copy number in transduced keratinocytes using a repeated transduction procedure. Finally, the intracellular tyrosine concentration was lowered by overexpression of tyrosinase converting tyrosine to dopaquinone. However, measured over a 24-hour period neither of these manipulations resulted in an increased phenylalanine uptake. These results suggest that other enzymes than GTP-CH, involved in BH4 synthesis and/or regeneration, can be rate-limiting in the genetically modified keratinocytes.
Christensen
Rikke
R
Department of Human Genetics, University of Aarhus, Aarhus, Denmark. rikkec@humgen.au.dk
Kolvraa
Steen
S
Jensen
Thomas G
TG
D027062:Amino Acid Transport System L; D000818:Animals; D002460:Cell Line; D006136:GTP Cyclohydrolase / Q000235:genetics* / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D010649:Phenylalanine / Q000378:metabolism*; D010651:Phenylalanine Hydroxylase / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013997:Time Factors
D027062:Amino Acid Transport System L; D010649:Phenylalanine; D010651:Phenylalanine Hydroxylase; D006136:GTP Cyclohydrolase