Phenotypic Reversion of Fair Hair upon Gene Therapy of the Phenylketonuria Mice

Phenylketonuria (PKU) (OMIM261600) is an autosomal recessive genetic disorder characterized by accumulation of the essential amino acid L-phenylalanine (L-Phe) in the body caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH; EC 1.14.16.1). Owing to attenuated biosynthesis of melanin due to systemic elevated L-Phe, hypopigmentation is one of the visible phenotypes of PKU. Such a phenotype can also be observed in PKU mice bearing the homozygous Pah-enu2 allele, where it was reported that restoration of hypopigmentation needs at least 5% of the PAH enzyme activity (Fang et al., 1994; Nagasaki et al., 1999; Viecelli et al., 2014). This phenotype was reversed in black 6 (C57Bl/6) PKU mice to wild-type level after hydrodynamic tail vein injection of a recombinant non-viral minicircle-based naked DNA vector expressing the murine Pah-cDNA from a liver-specific promoter. Young adult C57Bl/6 untreated wild-type and PKU mice carrying homozygously the Pah-enu2 allele are shown in Figure 1 before (middle), and 6 weeks after infusion of the minicircle vector MC.PKU20 (Viecelli et al., 2014). Fair-haired PKU mice started to darken after gene transfer and eventually became indistinguishable from wild-type. Changes from brown to black hair persisted from then on in minicircle vector–treated mice. Normalization of blood L-Phe concomitant with reversion of hypopigmentation in a dose-dependent manner can also be observed with other gene therapeutic vectors such as, for instance, after treatment of PKU with AAV2 serotype 8 (rAAV2/8-PKU5) to target liver or with rAAV2/1-mediated intramuscular expression of a complete L-Phe hydroxylating system (Ding et al., 2006, 2008).

FIG. 1.

Phenotypic reversion from brown to black coat color of treated phenylketonuria (PKU) (Pah-enu2) mouse (see text for details).

Acknowledgments

We thank the continuous support from the mass spectrometry units of the Divisions of Clinical Chemistry and Biochemistry and Newborn Screening, as well as Felix H. Sennhauser. Our work was supported by grants from the Children's Research Center Zurich, the Swiss National Science Foundation, the National Institutes of Health (Research Grant No. 1R01HD057033), and the Stiftung für wissenschaftliche Forschung der Universitat Zurich.