The overall goal of this project is to explore muscle-directed gene therapy as an alternative treatment for inborn errors of metabolism (IEM) detected by newborn screening. More specifically, the investigators will focus their efforts upon phenylalanine hydroxylase (PAH)-deficient Pahenu2 mice, a model of human phenylketonuria (PKU), and will evaluate expression of a phenylalanine (Phe) metabolizing system in muscle as a potential novel therapeutic approach to this disorder. For many IEM, disease pathology is due to effects of a circulating toxic metabolite rather than any direct tissue-specific effect from the enzyme deficiency. For example, PAH is primarily expressed in liver, but the pathology associated with PKU is entirely due to effects of circulating Phe;the liver itself is unaffected by PAH deficiency. Any treatment that achieves permanent reduction of blood Phe will effectively ameliorate the PKU phenotype. The investigators'hypothesized that expression of a robust Phe-metabolizing system in skeletal muscle will augment normal anabolic Phe uptake in muscle, effectively metabolize circulating Phe and correct hyperphenylalaninemia. They will explore two different Phe-metabolizing systems: coordinate expression in muscle of PAH along with the enzymes GTP cyclohydrolase 1 (GTPCH) and 6-pyruvoyltetrahydropterin synthase (PTPS), both elements of the tetrahydrobiopterin (BH4) synthetic pathway, to provide the BH4 cofactor necessary to support Phe hydroxylation or expression of phenylalanine ammonia lyase (PAL), a Phe-metabolizing enzyme that does not require any external cofactors.
In Specific Aim 1, the investigators will employ standard germline modification methods to develop transgenic mice that express a Phe metabolizing system throughout all skeletal muscle. These mice will be bred to Pahenu2 mice to yield progeny that lack liver PAH activity but express the Phe metabolizing system in muscle. Phe clearance will be assessed in these progeny.
In Specific Aim 2, they will separately employ either recombinant adenoassociated virus serotype 1 vectors (rAAV2/1) or naked plasmid DNA vectors delivered by hydrodynamic limb vein injection to induce expression of a Phe metabolizing system directly in hindlimb muscles of Pahenu2 mice. It is proposed that muscle expression of PAH along with elements of the BH4 synthetic pathway or of PAL alone will lead to correction of hyperphenylalaninemia in Pahenu2 mice.

Public Health Relevance

The overall goal of this project is to develop novel gene therapy approach for the treatment of inherited metabolic disorders that are detected at birth through newborn screening. The investigators will delineate muscle gene therapy as a new treatment approach to inherited metabolic disorders, and will focus specifically upon a mouse model of the human disorder phenylketonuria (PKU), one of the most common metabolic diseases.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Special Emphasis Panel (ZHD1-MRG-C (PA))
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Urv, Tiina K
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Oregon Health and Science University
Schools of Medicine
United States
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Viecelli, Hiu Man; Harbottle, Richard P; Wong, Suet Ping et al. (2014) Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver. Hepatology 60:1035-43
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