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 β-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 (BH₄), 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.