Expression of human phenylalanine hydroxylase activity in T lymphocytes of classical phenylketonuria children by retroviral-mediated gene transfer.

Classical phenylketonuria (PKU) is a metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. At present, T lymphocyte-directed gene therapy is the only means for which a safety record has been established. Thus, we investigated the applicability of this strategy to PKU gene therapy. We first looked for tetrahydrobiopterin (BH4) and dihydropteridine reductase (DHPR) activity, which are required for the phenylalanine hydroxylation reaction and BH4 regeneration, respectively, in T cells isolated from PKU children. We found that T cells contained a small amount of biopterin, but significant DHPR activity, and that the intracellular biopterin content could be increased by exogenous BH4 supplementation. Moreover, PKU T cells were capable of taking up phenylalanine efficiently and effluxing acquired tyrosine. Finally, a recombinant retrovirus containing the human PAH cDNA was constructed and used to transduce isolated PKU T cells. Viral-transduced T cells produced high levels of PAH activity as compared to control mock-infected T cells. These results indicate that T lymphocytes express all that is required for synthesizing/replenishing constituents of the phenylalanine hydroxylation reaction and expressing transduced phenylalanine hydroxylase cDNA.