The overall objective of this project is to investigate the efficacy of liver or muscle-directed gene therapy using recombinant adeno-associated virus (rAAV) vectors in the treatment of phenylketonuria (PKU) using, as a model, the phenylalanine hydroxylase (PAH) deficient Pahenu2 mouse. Recombinant adeno-associated virus has in recent studies shown promise as a gene delivery vector for both liver and muscle. We propose to compare the effects of muscle-directed vs. liver-directed rAAV-mediated gene therapy upon hyperphenylalaninemia in the Pahenu2 mouse. Restoration of liver enzyme activity is the most obvious approach for gene therapy of PKU. However, the disease pathophysiology associated with PKU is caused by circulating toxins, namely phenylalanine and its metabolites; PAH deficiency has no direct pathologic effect upon the liver. Therefore, any treatment approach that can effectively remove the toxic metabolites from the body could prevent disease-associated pathology. We have shown previously that PAH expression in muscle can lower serum phenylalanine levels if the enzyme is supplied with sufficient tetrahydrobiopterin (BH4), a required cofactor for PAH activity. However, attaining a sufficient continuous supply through exogenous administration of BH4 is very difficult and expensive. We propose two potential methods for avoiding this complication: engineering of skeletal muscle to express, along with PAH, components of the BH4 synthetic pathway or alternatively to induce muscle expression of phenylalanine ammonia lyase (PAL), an enzyme from yeast that requires no exogenous cofactors. In this project, we will first compare the ability of rAAV vectors to produce stable PAH expression in liver and muscle and assess the effect upon hyperphenyl-alaninemia in Pabenu2 mice. Secondly, we will develop germline modified mice that carry a muscle-specific transgene containing the PAH cDNA and elements of the BH4 synthetic pathway (GTP cyclohydrolase I (GTPCH) or GTPCH and pyruvoyltetrahydropterin synthase (PTPS)) in an attempt to induce both constitutive PAH expression and BH4 synthesis. We will breed these animals to Pahenu2 mice to determine whether muscle PAH expression together with BH4 synthesis can correct hyperphenylalaninemia in PKU. Finally, we will investigate PAL expression induced either by germline modification or rAAV-mediated gene transfer in Pahenu2 mouse muscle, as a possible treatment of hyperphenylalaninemia. The ultimate goal of our research is the development of effective and safe gene therapy for phenylketonuria.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK059371-01
Application #
6317786
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Mckeon, Catherine T
Project Start
2001-07-01
Project End
2003-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
1
Fiscal Year
2001
Total Cost
$148,960
Indirect Cost
Name
Oregon Health and Science University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Harding, Cary O; Winn, Shelley R; Gibson, K Michael et al. (2014) Pharmacologic inhibition of L-tyrosine degradation ameliorates cerebral dopamine deficiency in murine phenylketonuria (PKU). J Inherit Metab Dis 37:735-43
Hamman, Kelly J; Winn, Shelley R; Harding, Cary O (2011) Hepatocytes from wild-type or heterozygous donors are equally effective in achieving successful therapeutic liver repopulation in murine phenylketonuria (PKU). Mol Genet Metab 104:235-40
Harding, Cary O; Blau, Nenad (2010) Advances and challenges in phenylketonuria. J Inherit Metab Dis 33:645-8
Rebuffat, Alexandre; Harding, Cary O; Ding, Zhaobing et al. (2010) Comparison of adeno-associated virus pseudotype 1, 2, and 8 vectors administered by intramuscular injection in the treatment of murine phenylketonuria. Hum Gene Ther 21:463-77
Arnold, Georgianne L; Van Hove, Johan; Freedenberg, Debra et al. (2009) A Delphi clinical practice protocol for the management of very long chain acyl-CoA dehydrogenase deficiency. Mol Genet Metab 96:85-90
Ding, Zhaobing; Harding, Cary O; Rebuffat, Alexandre et al. (2008) Correction of murine PKU following AAV-mediated intramuscular expression of a complete phenylalanine hydroxylating system. Mol Ther 16:673-81
Harding, Co (2008) Progress toward cell-directed therapy for phenylketonuria. Clin Genet 74:97-104
Harding, C O; Gillingham, M B; Hamman, K et al. (2006) Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria. Gene Ther 13:457-62
Hamman, Kelly; Clark, Heather; Montini, Eugenio et al. (2005) Low therapeutic threshold for hepatocyte replacement in murine phenylketonuria. Mol Ther 12:337-44
Harding, Cary O; Neff, Mark; Wild, Krzysztof et al. (2004) The fate of intravenously administered tetrahydrobiopterin and its implications for heterologous gene therapy of phenylketonuria. Mol Genet Metab 81:52-7

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