Engineering a Supplemental Phenylalanine Metabolic Pathway to Prevent mPKU

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DESCRIPTION (provided by applicant): The Hunter lab proposes to develop genetically modified cells to overproduce Phenylalanine Hydroxylase (PAH). This enzyme is frequently mutated and completely or partially inactive in persons with phenylketonuria (PKU). Without this enzyme, high levels of phenylalanine and its metabolites build up and through an unknown pathway lead to profound and irreversible mental retardation, seizures, and reduced growth. The current therapy is nutritional supplements and dietary restriction limiting the intake of phenylalanine. Although difficult, this therapy is recommended for the life of the individual. While the combination of newborn screening and dietary therapy has been extremely successful in preventing severe mental retardation in PKU patients, several inadequacies exist. Some treated children still suffer mild cognitive defects. Main inhibitors of patient compliance include the foul taste, expense, and restrictiveness of the diet. Because the diet is very limited and distasteful, many pregnant women with PKU cannot tolerate it and consequently cannot control their phenylalanine levels. The consequence is termed maternal PKU syndrome. Maternal PKU syndrome has only emerged within the last 15-20 years as the first generation of women to receive dietary treatment as children reach child-bearing age. These women have normal fertility. However, prior to the dietary therapy, most PKU patients had profound cognitive problems and did not reproduce. In maternal PKU syndrome, the child does not necessarily suffer from the metabolic error. Instead, the maternal PKU has teratogenic effects on the fetus. Maternal PKU affects the fetal brain, growth, facial morphology, and cardiac development. The objective of this proposal is to develop and evaluate cell lines for their ability to over-express PAH and to process phenylalanine. These cells will then be encapsulated within polymeric microspheres. The microspheres isolate these cells from the immune system. Encapsulated cells will be injected intraperitoneally into PKU model mice. Mice will be evaluated for changes in their ability to metabolize phenylalanine before and after implantation. In addition, mice will be mated and the physical and mental characteristics of the pups will be evaluated. Furthermore, mice will be injected at different time points during pregnancy to evaluate the effects of "boosting" the phenylalanine metabolic pathway with the encapsulated cells at different stages of pregnancy on the pups.