SULM – Schweizerische Union für Labormedizin | Union Suisse de Médecine de Laboratoire | Swiss Union of Laboratory Medicine

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Z Ding 1 , B Thöny 1

1Division of Clinical Chemistry, Department of Pediatrics, University of Zürich, Switzerland

Phenylketonuria (PKU) or hyperphenylalaninemia is an autosomal recessive disorder caused by deficiency of the hepatic phenylalanine hydroxylase (PAH) that clears plasma phenylalanine by conversion to tyrosine using molecular oxygen and tetrahydrobiopterin (BH4) as cofactor. It is a metabolic disease which can potentially be corrected by heterologous gene expression and degradation of phenylalanine in liver or another organ than liver. However, for efficient degradation of phenylalanine in tissues that do not produce sufficient BH4 cofactor, heterologous expression of PAH together with BH4 biosynthetic genes is required. Here we constructed various monocistronic and polycistronic gene expression cassettes, the latter by the use of different ‘internal ribosome entry sites’ (IRES) between the mouse cDNAs for PAH and BH4-biosynthetic genes. Expression from the various promoters with additional post-transcriptional regulatory elements was verified by transient lipofectamine-transfection into COS-1 cells, followed by assaying specific enzyme activities. We then generated hybrids consisting of recombinant vector plasmids AAV type 2 (AAV2) with Cap proteins from either type 1 for muscle-directed gene transfer (called AAV2/1), or Cap proteins type 2 or 8 for liver-directed gene transfer (AAV2/2 or AAV2/8), with titers of up to 1x10E11 vector genomes per ml. Upon administration of recombinant AAV2/2 to PAH-deficient mice via the tail vein, we observed a decrease in plasma phenylalanine in the first weeks after injection. Further studies are needed to clarify whether metabolic long-term correction in this PKU mouse model can be achieved with recombinant AAV and/or SIN lentiviral constructs harboring the phenylalanine degradation activity for liver or muscle gene therapy.

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