Use of an adeno-associated virus serotype Anc80 to provide durable cure of phenylketonuria in a mouse model

Abstract

Phenylketonuria (PKU) is the most common inborn error of metabolism of the liver, and results from mutations of both alleles of the phenylalanine hydroxylase gene (PAH). As such, it is a suitable target for gene therapy via gene delivery with a recombinant adeno-associated virus (AAV) vector. Here we use the synthetic AAV vector Anc80 via systemic administration to deliver a functional copy of a codon-optimized human PAH gene, with or without an intron spacer, to the Pah^enu2 mouse model of PKU. Dose-dependent transduction of the liver and expression of PAH mRNA were present with both vectors, resulting in significant and durable reduction of circulating phenylalanine, reaching near control levels in males. Coat color of treated Pah^enu2 mice reflected an increase in pigmentation from brown to the black color of control animals, further indicating functional restoration of phenylalanine metabolism and its byproduct melanin. There were no adverse effects associated with administration of AAV up to 5 × 10¹² VG/kg, the highest dose tested. Only minor and/or transient variations in some liver enzymes were observed in some of the AAV-dosed animals which were not associated with pathology findings in the liver. Finally, there was no impact on cell turnover or apoptosis as evaluated by Ki-67 and TUNEL staining, further supporting the safety of this approach. This study demonstrates the therapeutic potential of AAV Anc80 to safely and durably cure PKU in a mouse model, supporting development for clinical consideration.

Keywords: Anc80; adeno-associated virus (AAV) vector; gene therapy; inborn error of metabolism; phenylketonuria.

FIGURE 1

A, Western blot analysis of phenylalanine hydroxylase gene (PAH) expression following plasmid transfection in HuH7 or Hepa1‐6 cell lines with four construct variants of human PAH. B, Relative protein expression levels normalized to a mock control for transfection. Green fluorescent protein (GFP), mock control. C, PAH mRNA levels following transduction with adeno‐associated virus (AAV) with and without the MVM intron spacer in HuH7 cells. Statistical significance was assessed by unpaired t test, **P < .01

FIGURE 2

Mice dosed with increasing levels of Anc80‐green fluorescent protein (GFP) showed dose dependent transduction and vector expression. A, There were no dose‐related variations in body weights over the course of the study within genotypes regardless of vector dose administered. Increasing doses of Anc80‐GFP resulted in dose‐dependent increases in, B, adeno‐associated virus (AAV) genomes and C, GFP transcripts present in the livers of Pah^enu2 mice. Males showed enhanced responsiveness relative to females in both parameters, with a similar trend present in the wt mice tested at the mid dose of 5 × 10¹² VG/kg. D, There was high transduction frequency in the livers at all dose levels tested based on IHC of GFP in representative fixed sections, leaving only incremental gains with increasing doses. E, Immunohistochemistry micrographs of GFP staining in representative sections of livers from male and female mice in each dose group. Dose‐dependent increases in transduction frequency were present in Pah^enu2 mice, but were more pronounced in females due to the lower percentage of transduced hepatocytes at the low and mid doses compared to males. Staining in wt mice at the mid dose was similar to that in the Pah^enu2 mice, with the same trend toward increased transduction in males. One‐way analysis of variance (ANOVA) with multiple comparisons and Bonferroni correction were used to assess statistical significance, P < .05 (*) .01 (**), .001 (***), or .0001 (****)

FIGURE 3

Expression of phenylalanine hydroxylase gene (PAH) corrected the phenylketonuria (PKU) phenotype in Pah^enu2 mice. The high dose of 5 × 10¹² VG/kg Anc80‐coPAH and Anc80‐int‐coPAH was associated with acute and sustained decreases in circulating phenylalanine levels in males, A, and females, B, which approached wt levels in males with both PAH constructs. Only negligible benefit was indicated at the lower dose of 5 × 10¹¹ VG/kg. C, Wild‐type animals (first column) had much darker coat color than untreated Pah^enu2 mice (second column), and restored metabolism of phenylalanine caused a phenotypic switch back to darker coat color in treated male (top row) and female (bottom row) mice which was dose‐dependent. D, Adeno‐associated virus (AAV) genomes in the liver relative to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) showed similar dose‐dependent increases between the 5 × 10¹¹ VG/kg and 5 × 10¹² VG/kg levels. E, PAH mRNA showed corresponding increases between dose levels, with slightly higher transcription with Anc80‐coPAH. In both analyses, results were negligible for wt animals and Pah^enu2 mice dosed with saline. One‐way analysis of variance (ANOVA) with multiple comparisons and Dunnett's correction were used to assess statistical significance, P < .05 (*) .01 (**), .001 (***), or .0001 (****)

FIGURE 4

Adeno‐associated virus (AAV) transduction and expression of phenylalanine hydroxylase gene (PAH) was well tolerated in all animals. A, There were no significant variations in body weights over the course of the study within genotypes regardless of vector dose administered. Serum liver enzymes indicated only minor effects on hepatocytes, with Anc80‐int‐coPAH being associated with a minor elevation in, B, ALT at the high dose relative to the untreated Pah^enu2 control mice, and no effects of either Anc80 construct on, C, AST, or D, ALP at week 12

FIGURE 5

Anc80‐adeno‐associated virus (AAVs) were not associated with changes in cell turnover or apoptosis. A, Percentage of cells that were positive for Ki‐67 staining were similarly low in all groups tested, showing no increased cell turnover resulting from transduction with either AAV. B, Percentage of cells undergoing apoptosis as indicated by TUNEL positivity was also similarly low in all groups tested. In both analyses, results were similar for treated groups, wt animals, and Pah^enu2 mice dosed with saline. One‐way analysis of variance (ANOVA) with multiple comparisons and Dunnett's correction were used to assess statistical significance, P < .05 (*) P < .005 (**)