Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria

Abstract

Novel recombinant adeno-associated virus vectors pseudotyped with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pah enu2 mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pah enu2 mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.572.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liverdirected gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism.

Glycomacropeptide, a low-phenylalanine protein isolated from cheese whey, supports growth and attenuates metabolic stress in the murine model of phenylketonuria

“…The animal facilities and protocols reported were approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee. A PKU mouse colony was established using the murine model of PAH deficiency, the Pah enu mouse, on a C57Bl/6 background (20,32). PKU heterozygous mice were bred to produce PKU homozygous mice and WT control mice.…”

The Role of Hepatocyte Hemojuvelin in the Regulation of Bone Morphogenic Protein-6 and Hepcidin Expression in Vivo

“…After verification by sequencing, Hjv cDNA was subcloned into an AAV2/8 vector containing a strong liver-specific promoter (LSP). The LSP is a combination of two copies of a human ␣1-microglobulin/ bikunin enhancer and the promoter from the human thyroid hormone-binding globulin gene (21)(22)(23)(24). Vectors were generated at the Gene Therapy Center Virus Vector Core Facility, University of North Carolina at Chapel Hill by co-transfecting 293 cells with AAV2/8-Hjv vector, the AAV packaging plasmid and pAdHelper.…”

“…AAV2/8 Vector Specifically Introduced Hjv into HepatocytesTo determine the role of hepatocyte HJV in hepcidin expression, we used AAV2/8 vector that has a strong liver-specific enhancer/promoter (21)(22)(23)(24), as a tool to specifically introduce exogenous mouse Hjv cDNA into the Hjv Ϫ/Ϫ murine hepatocytes. Previous studies demonstrate that this vector is able to specifically target the gene of interest into the hepatocytes and that the expression of the introduced gene is evenly distributed in hepatocytes throughout the liver (22,29).…”

In search of proof‐of‐concept: gene therapy for glycogen storage disease type Ia

“…AAV vector-mediated gene therapy has effectively treated GSD-Ia and other diseases requiring persistent transgene expression in the available mouse models, including hemophilia, phenylketonuria, tyrosinemia, and other metabolic disorders, due to relative long-term stability of episomal vector genomes in hepatocytes (Chen et al 2000;Harding et al 2006;Herzog et al 1999;Koeberl et al 2006). A number of AAV serotypes are available that transduce the liver with high efficiency in mice, and several of these serotypes have reproducibly transduced the liver in canine genetic disease models, including AAV serotype 2 (AAV2) vectors that were pseudotyped as AAV serotype 8 (AAV2/8) and AAV2/9 (Koeberl et al 2008;Sarkar et al 2006).…”