The fate of intravenously administered tetrahydrobiopterin and its implications for heterologous gene therapy of phenylketonuria.

Tetrahydrobiopterin (BH(4)) is a required cofactor for the enzymatic activity of phenylalanine hydroxylase (PAH) and is synthesized de novo from GTP in several tissues. Heterologous expression of PAH in tissues other than liver is a potential novel therapy for human phenylketonuria that is completely dependent upon BH(4) supply in the PAH-expressing tissue. Previous experiments with liver PAH-deficient transgenic mice that expressed PAH in skeletal muscle demonstrated transient correction of hyperphenylalaninemia only with hourly parenteral BH(4) administration. In this report, the fate of intravenously administered BH(4) is examined. The conclusions are that (1) BH(4) administered intravenously is rapidly taken up by liver and kidney, and (2) uptake of BH(4) into muscle is relatively low. The levels of BH(4) achieved in skeletal muscle following IV injection are only 10% of the amount expected were BH(4) freely and equally distributed across all tissues. The half-life of BH(4) in muscle is approximately 30 min, necessitating repeated injections to maintain muscle BH(4) content sufficient to support phenylalanine hydroxylation. The efficacy of heterologous muscle-directed gene therapy for the treatment of PKU will likely be limited by the BH(4) supply in PAH-expressing muscle.

Expression of phenylalanine hydroxylase (PAH) in erythrogenic bone marrow does not correct hyperphenylalaninemia in Pah(enu2) mice.

BACKGROUND: Treatment of many inherited liver enzyme deficiencies requires the removal of toxic intermediate metabolites from the blood of affected individuals. We propose that circulating toxins can be adequately cleared and disease phenotype influenced by enzyme expressed in tissues other than the liver, such as bone marrow. Our specific hypothesis was that phenylalanine hydroxylase (PAH) expressed in bone marrow would lower blood phenylalanine levels in hyperphenylalaninemic Pah(enu2) mice, a model of human phenylketonuria (PKU). METHODS: Germline-modified marrow PAH-expressing mice were developed using a transgene that contained the mouse liver PAH cDNA under the transcriptional control of a human beta-globin promoter. Marrow PAH-expressing mice were bred to Pah(enu2) mice to generate progeny that lacked liver PAH activity but expressed PAH in bone marrow. RESULTS: Marrow PAH expression did not affect the health, function, or reproductive capacity of transgenic animals. Hyperphenylalaninemia persisted in transgenic Pah(enu2) homozygous mice despite PAH activity in marrow lysates, and was not altered following supplementation with tetrahydrobiopterin (BH(4)), a required cofactor for PAH. PAH activity measured in intact marrow cells was significantly lower than in marrow lysates; no such difference was measured in isolated hepatocytes vs. liver homogenate. CONCLUSIONS: Marrow PAH expression did not correct hyperphenylalaninemia in Pah(enu2) mice. Phenylalanine clearance may have been limited by the natural perfusion rate of the marrow compartment, by insufficient PAH expression in marrow, or by other cellular factors affecting phenylalanine metabolism in intact marrow cells. Differences in PAH activity measured in intact marrow cells vs. cell lysates suggest that hepatocytes and PAH-expressing marrow cells are fundamentally different in their ability to metabolize phenylalanine. The efficacy of bone-marrow-directed gene therapy as a metabolic sink in the treatment of phenylketonuria may be limited, although further experiments with greater marrow PAH expression levels will be necessary to definitively prove this conclusion.