Intravenous non-viral gene therapy for phenylketonuria (PKU)

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DESCRIPTION (provided by applicant): The overall goal of this project is to develop a safe, effective, non-viral, muscle-directed gene therapy approach to the treatment of phenylketonuria (PKU), a relatively common inborn error of metabolism. Contemporary dietary therapy for PKU, although preventing the major manifestations of the disease (seizures, microcephaly, mental retardation), requires life-long commitment to an unpalatable and expensive diet. Dietary noncompliance is common. An effective, safe, permanent treatment for PKU that does not rely upon dietary manipulation is desirable. The pathology associated with PKU is caused by the accumulation of phenylalanine (Phe); any method that successfully reduces Phe levels will ameliorate the PKU phenotype. We propose that PAH activity in skeletal muscle will act as a metabolic sink to clear Phe from the circulation. To this end, we will evaluate a novel intravenous hydrodynamic DNA transfer method to express the enzyme phenylalanine hydroxylase (PAH) in skeletal muscle of hyperphenylalaninemic Pahenu2 mice, a model of human PKU. PAH requires the unconjugated pterin, tetrahydrobiopterin (BH4), as a cofactor. Skeletal muscle contains little BH4. Preliminary data demonstrate that expression of elements of the BH4 synthetic pathway in skeletal muscle is required to maintain sufficient BH4 and support Phe hydroxylation. Our specific hypothesis is that coordinated expression of PAH along with the BH4-synthetic enzymes, GTP cyclohydrolase (GTPCH) and 6-pyruvoyltetrahydropterin synthase (PTPS) in skeletal muscle will lead to sufficient BH4 production, sustained PAH activity, and a physiologically significant decrease in blood Phe levels in Pahenu2 mice. Our plan is to express the PAH, GTPCH, and PTPS cDNAs in Pahenu2 skeletal muscle by delivering naked plasmid DNA intravenously using a hydrodynamic limb vein technique, a procedure that is equally effective in small and large research animals. We will evaluate blood Phe levels, Phe tolerance and physical well-being of the mice before and after injection. The amount and duration of gene expression will also be evaluated. Our experiments will also compare gene expression from a triple-cistronic plasmid vector vs. co-administration of 3 separate plasmid vectors encoding single genes. These preclinical trials will form the basis for the development of future clinical trials in humans with PKU.