Metabolic engineering as therapy for inborn errors of metabolism – development of mice with phenylalanine hydroxylase expression in muscle

Harding, CO; Wild, Krzysztof; Chang, David; Messing, Albee; Wolff, J.

doi:10.1038/sj.gt.3300653

Cited by 37 publications

(37 citation statements)

References 23 publications

(28 reference statements)

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“…The murine Pah-cDNA was amplified by PCR from plasmid pBlueMCK-bgmPAH (kindly provided by CO Harding 43 ) with primers containing the terminal restriction sites BamHI and EcoRI and cloned into pUC18 to generate pHSY106. The Pah-cDNA fragment was then excised by BamHI and EcoRI, filled-in by Klenow polymerase, and cloned into the PmlI site of the AAV2 vector plasmid (provided by H Bü eler 17 ) to generate plasmid pAAV2-PKU-5.…”

Section: Recombinant Aav Construction and Productionmentioning

confidence: 99%

Administration-route and gender-independent long-term therapeutic correction of phenylketonuria (PKU) in a mouse model by recombinant adeno-associated virus 8 pseudotyped vector-mediated gene transfer

2005

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Phenylketonuria (PKU) is an inborn error of metabolism caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH) which leads to high blood phenylalanine (Phe) levels and consequent damage of the developing brain with severe mental retardation if left untreated in early infancy. The current dietary Phe restriction treatment has certain clinical limitations. To explore a long-term nondietary restriction treatment, a somatic gene transfer approach in a PKU mouse model (C57Bl/6-Pah enu2 ) was employed to examine its preclinical feasibility. A recombinant adenoassociated virus (rAAV) vector containing the murine Pah-cDNA was generated, pseudotyped with capsids from AAV serotype 8, and delivered into the liver of PKU mice via single intraportal or tail vein injections. The blood Phe concentrations decreased to normal levels (p100 mM or 1.7 mg/dl) 2 weeks after vector application, independent of the sex of the PKU animals and the route of application. In particular, the therapeutic long-term correction in females was also dramatic, which had previously been shown to be difficult to achieve. Therapeutic ranges of Phe were accompanied by the phenotypic reversion from brown to black hair. In treated mice, PAH enzyme activity in whole liver extracts reversed to normal and neither hepatic toxicity nor immunogenicity was observed. In contrast, a lentiviral vector expressing the murine Pah-cDNA, delivered via intraportal vein injection into PKU mice, did not result in therapeutic levels of blood Phe. This study demonstrates the complete correction of hyperphenylalaninemia in both males and females with a rAAV serotype 8 vector. More importantly, the feasibility of a single intravenous injection may pave the way to develop a clinical gene therapy procedure for PKU patients.

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Section: Recombinant Aav Construction and Productionmentioning

confidence: 99%

Administration-route and gender-independent long-term therapeutic correction of phenylketonuria (PKU) in a mouse model by recombinant adeno-associated virus 8 pseudotyped vector-mediated gene transfer

2005

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“…To assess whether heterologous expression of Pah in muscle would lead to degradation of phenylalanine, the Pah gene was put under control of the muscle creatine kinase promoter, and was constitutively expressed in skeletal and cardiac muscle of transgenic mice generated by classical microinjection of eggs [50]. Breeding such mice to the Pah enu2 strain for homozygosity resulted in offspring lacking PAH activity in the liver but expressing Pah in the skeletal muscle.…”

Section: Heterologous Non-liver Gene Therapy For Pkumentioning

confidence: 99%

State-of-the-art 2003 on PKU gene therapy

Ding

Harding

Thöny

2004

Molecular Genetics and Metabolism

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Phenylketonuria (or PKU) is a well-known and widespread genetic disease for which many countries perform newborn screening, and life-long dietary restriction is still the ultimate and effective therapy. However, the diet is complicated, unpalatable, and expensive. The long-term effects of diet discontinuation in adults, except for the serious adverse effects of maternal hyperphenylalaninemia upon the developing fetus, have not been systematically studied, but congnitive decline and neurologic abnormalities have been anecdotally reported. Thus, alternative approaches for PKU therapy, including gene therapy, must be further explored. Here we summarize past present nonviral and viral gene transfer approaches, both in vitro studies and preclinical animal trials, to delivering the PAH gene into liver or other organs as potential alternatives to life-long phenylalanine-restricted dietary theraphy.

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“…The success of heterologous gene therapy for PKU using PAH is dependent upon BH 4 supply to the target tissue. We have previously demonstrated reduced serum phenylalanine levels in muscle PAH-expressing, liver PAH-deficient mice when BH 4 is administered repetitively and in large amounts [3]. The goal of the experiments presented here was to further our understanding of the fate of exogenously administered BH 4 .…”

Section: Discussionmentioning

confidence: 99%

The Fate of Intravenously Administered Tetrahydrobiopterin and Its Implications for Heterologous Gene Therapy of Phenylketonuria

Harding¹,

Neff²,

Wild³

et al. 2002

Chemistry and Biology of Pteridines and Folates

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Tetrahydrobiopterin (BH 4 ) is a required cofactor for the enzymatic activity of phenylalanine hydroxylase (PAH) and is synthesized de novo from GTP in several tissues. Heterologous expression of PAH in tissues other than liver is a potential novel therapy for human phenylketonuria that is completely dependent upon BH 4 supply in the PAH-expressing tissue. Previous experiments with liver PAH-deficient transgenic mice that expressed PAH in skeletal muscle demonstrated transient correction of hyperphenylalaninemia only with hourly parenteral BH 4 administration. In this report, the fate of intravenously administered BH 4 is examined. The conclusions are that (1) BH 4 administered intravenously is rapidly taken up by liver and kidney, and (2) uptake of BH 4 into muscle is relatively low. The levels of BH 4 achieved in skeletal muscle following IV injection are only 10% of the amount expected were BH 4 freely and equally distributed across all tissues. The half-life of BH 4 in muscle is approximately 30 min, necessitating repeated injections to maintain muscle BH 4 content sufficient to support phenylalanine hydroxylation. The efficacy of heterologous muscledirected gene therapy for the treatment of PKU will likely be limited by the BH 4 supply in PAHexpressing muscle.

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Metabolic engineering as therapy for inborn errors of metabolism – development of mice with phenylalanine hydroxylase expression in muscle

Cited by 37 publications

References 23 publications

Administration-route and gender-independent long-term therapeutic correction of phenylketonuria (PKU) in a mouse model by recombinant adeno-associated virus 8 pseudotyped vector-mediated gene transfer

Administration-route and gender-independent long-term therapeutic correction of phenylketonuria (PKU) in a mouse model by recombinant adeno-associated virus 8 pseudotyped vector-mediated gene transfer

State-of-the-art 2003 on PKU gene therapy

The Fate of Intravenously Administered Tetrahydrobiopterin and Its Implications for Heterologous Gene Therapy of Phenylketonuria

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