Correction of murine PKU following AAV-mediated intramuscular expression of a complete phenylalanine hydroxylating system

Phenylketonuria (PKU) caused by phenylalanine hydroxylase (PAH) deficiency leads to toxic accumulation of phenylalanine (Phe). PAH is predominantly expressed in liver and its activity requires a supply of tetrahydrobiopterin (BH4) cofactor, but we propose that expression of a complete Phe hydroxylating system (PAH plus BH4 synthetic enzymes) in skeletal muscle will lead to therapeutic reduction of blood Phe levels in Pah(enu2) mice, a model of human PKU. In order to test this hypothesis, we first developed transgenic Pahenu2 mice that lack liver PAH activity but coexpress, in their skeletal muscle, PAH and guanosine triphosphate cyclohydrolase I (GTPCH). The latter is responsible for the committing enzymatic step in BH4 biosynthesis. Despite sufficient muscle enzyme expression, these mice remained hyperphenylalaninemic, thereby suggesting that expression of additional BH4 synthetic enzymes would be necessary. A recombinant triple-cistronic adeno-associated virus-2 (AAV2) pseudotype 1 vector expressing PAH along with GTPCH and 6-pyruvoyltetrahydrobiopterin synthase (PTPS), the next step in BH4 synthesis, was generated. Injection of this vector into the gastrocnemius muscles of Pah(enu2) mice led to stable and long-term reduction of blood Phe and reversal of PKU-associated coat hypopigmentation. We propose that muscle-directed gene therapy will be a viable alternative treatment approach to PKU and other inborn errors of metabolism.