Cerreto, Monica (2010) Phenylketonuria: from the characterization of new mutations to the correction of the pathologic phenotype by a helper dependent adenoviral vector expressing PAH. [Tesi di dottorato] (Unpublished)

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Background: Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism and is caused by defective activity of phenylalanine hydroxylase (PAH), the enzyme that converts phenylalanine (Phe) to tyrosine (Tyr). The biochemical consequences of this deficit are the accumulation of Phe and the decrease of Tyr levels in the blood, which results in a strong imbalance of the Phe/Tyr ratio. If untreated, PKU leads to a severe neurologic impairment, whose aetiology is still unclear. At present, PKU therapy is based on dietary restriction of Phe that is very challenging in practice because it requires life-long adherence, it is unpleasant in taste, involves a risk of nutritional deficiencies, and has a substantial psychosocial burden. Consequently, a more effective cure for PKU is desirable. To this aim, we explored a gene therapy approach using a helper-dependent adenoviral vector expressing PAH. Methods: We produced a helper-dependent adenoviral (HD-Ad) vector expressing the h-PAH cDNA and administered it to 3-week-old PKU mice. Mice were subjected to biochemical analyses (Phe/Tyr determination, PAH activity), behavioural tasks (Morris Water Maze, Fear Conditioning), and synaptic plasticity studies (long-term potentiation, LTP) to evaluate the rescue of their pathologic phenotype. We also studied the expression of N-methyl-D-aspartic acid (NMDA) and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits. Results: We obtained complete normalization of serum Phe levels in HD-Ad-treated mice 2 weeks after the administration of the therapeutic vector, and a reversal of PKU-associated coat hypopigmentation. These effects were long lasting because the decrease of Phe and the increase of Tyr lasted throughout the observation period (6 months). Moreover, we observed a strong deficit in spatial learning and memory in untreated PKU mice that was completely reversed in HD-Ad-treated mice, at both 2 and 6 months of treatment. Studies of synaptic plasticity mirrored this recovery, since we found that LTP was impaired in untreated PKU mice but was restored in gene therapy-treated mice. Finally, we found an imbalance in the expression of the NMDA receptors NR2A and NR2B and of the α amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors Glu1 and Glu2/3 in PKU mice that improved after gene therapy. Conclusion: Our data suggest that HD-Ad-mediated gene therapy can be a promising approach to treat PKU because it allows a long-term correction of the biochemical and behavioural phenotypes in PKU mice. Moreover, the biochemical correction mediated by our treatment also improved LTP activity and AMPA and NMDA receptor expression.

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