Therapy of PKU by AAV-1 based transfer of PAH and BH4-cofactor genes into skeletal muscle. B. Thony¹, C.O. Harding², A. Rebuffat¹, L. Elzazouk¹, J.A. Wolff³, Z. Ding¹. 1) Dept Ped, Div Clin Chem/Biochem, Univ Zurich, Zurich, Switzerland; 2) Dept Ped, Dept Molec/Med Genet, Oregon Health/Science Univ, Portland, OR, USA; 3) Dept Ped, Dept Med Genet, Univ Wisconsin, Madison, WI, USA.
   Phenylketonuria (PKU) is caused by an autosomal recessive deficiency of the hepatic phenylalanine hydroxylase (PAH) leading high serum Phe and irreversible damage of the brain. PAH catalyzes the hydroxylation of Phe to tyrosine with tetrahydrobiopterin (BH4) as cofactor. BH4 biosynthesis requires the consecutive action of the enzymes GTPCH and PTPS, with dihydroneopterin triphosphate as the first intermediate. Here we aimed at expressing the PAH system in skeletal muscle to degrade serum Phe, as this tissue is abundant, easy accessible, and persistent due to its post-mitotic nuclei. However, BH4 is scarce in skeletal muscle as the cofactor-synthesizing enzyme GTPCH is absent in muscle tissue, and PTPS is expressed at low levels. We first demonstrated that transgenic PKU mice that had no liver PAH and expressed coordinately PAH along with GTPCH in skeletal muscle tissue accumulated dihydroneopterin triphosphate and remained hyperphenylalaninemic unless synthetic BH4-cofactor was supplied by intraperitoneal injections. Thus, PTPS activity is definitely limiting in skeletal muscle to synthesize sufficient BH4 and to support Phe hydroxylation. A recombinant triple-cistronic AAV2-based pseudotype 1 vector expressing PAH along with the two cDNA-genes for BH4 biosynthesis, GTPCH and PTPS, was then generated. Upon single injections of 3.5x10e12 recombinant triple-cistronic AAV2/1 vector particles into each of the gastrocnemius muscles of the hind legs of the PKU mouse model Pah-enu2 resulted in long-term clearance of blood Phe, including complete phenotypic reversion. A similar therapeutic effect was achieved when a combination of two AAV2/1 vectors expressing individually PAH and GTPCH-PTPS were co-injected into the same hind leg muscles. As a control, an AAV2/1-vector expressing only PAH with GTPCH was not therapeutic. This non-invasive application is the basis to develop an efficient therapy for PKU using a triple-cistronic gene transfer into skeletal muscle.