Long-Term Enzymatic and Phenotypic Correction in the Phenylketonuria Mouse Model by Adeno-Associated Virus Vector-Mediated Gene Transfer

Abstract

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH). The accumulation of phenylalanine leads to severe mental and psychomotor retardation, and hypopigmentation of skin and hair. Low-phenylalanine diet therapy can prevent irreversible damage if instituted from birth. However, poor compliance with the strict lifelong dietary therapy leads to various neurologic and behavioral problems. To develop a safe and promising gene therapy method for PKU, we investigated whether a recombinant adeno-associated virus could be used as a PAH gene transfer vector to reduce the excessive phenylalanine level in the PKU mouse model. A recombinant adeno-associated virus vector encoding the human PAH gene (rAAV-hPAH), driven by EF1-␣ promoter, was infused into PAH-deficient mice, Pah enu2 , via the hepatic portal vein. Two weeks after injection, the plasma phenylalanine level dramatically decreased to 360 M in male PKU mice, accompanied by the coat color changing to black. The mean plasma phenylalanine level of untreated PKU mice was 1800 M. The PAH enzyme activities of treated mice increased to 10 -17% of wild-type mice. No signs of liver toxicity were observed after gene transfer. The biochemical and phenotypic corrections were sustained for up to 25 wk (25-wk detection period). In contrast, the treatment was less effective in female PKU mice. These results indicate that recombinant adenoassociated virus vector-mediated gene therapy can be a useful therapeutic candidate for patients with PKU. Further studies are needed to clarify the differences in PKU pathogenesis in males and females, and to explore alternative administration routes besides hepatic portal vein injection. Abbreviations PKU, phenylketonuria PAH, phenylalanine hydroxylase AAV, adeno-associated virus rAAV-hPAH, recombinant adeno-associated virus vector encoding human phenylalanine hydroxylase cDNA BH 4 , tetrahydrobiopterin RT-PCR, reverse-transcriptase polymerase chain reaction TLC, thin layer chromatography PKU (MIM 261600) is an autosomal recessive metabolic disorder caused by a number of mutations, primarily singlebase substitutions, in the PAH gene, located on chromosome 12q22-24.1 (1, 2). This disorder is characterized by the disruption of phenylalanine metabolism due to the lack of phenylalanine hydroxylase activity. PAH (L-phenylalanine-4-monooxygenase, EC 1.14.16.1), predominantly expressed in liver, catalyzes the conversion of the essential amino acid phenylalanine into tyrosine using BH 4 as a cofactor (3, 4). The loss of this enzymatic activity results in the accumulation of phenylalanine and its abnormal derivatives in the blood and other tissues. Excessive blood phenylalanine concentration causes severe mental and psychomotor retardation from toxicity to the CNS, although the exact mechanism is not well known, and other clinical symptoms such as hypopigmentation of skin, hair, and eyes, growth failure, and "mousy" odor (5, 6).These symptoms can be prevented by a strict, lifelong die...

Administration-route and gender-independent long-term therapeutic correction of phenylketonuria (PKU) in a mouse model by recombinant adeno-associated virus 8 pseudotyped vector-mediated gene transfer

“…When comparing the Phe concentrations of male and female PKU mice, we found that the Phe concentrations in females were slightly but not significantly higher than in males (males: 16957385 mM, n ¼ 21; females: 18667256 mM, n ¼ 22; P-value 0.0997). This reflects to some extent the disagreement published by Mochizuki et al 27 in comparison with the publication by Oh et al 28 with regard to sex-dependent blood Phe concentrations in PKU mice (see also discussion below).…”

“…In summary, we found after injection of 5 Â 10 12 vector genomes per mouse approximately 10 3 rAAV copies per transduced hepatocyte which is comparable to Nakai et al 25 that reported a transduction rate of around 620 copies in liver cells for 7.2 Â 10 12 vector genomes per injected mouse. Unfortunately, transduction rates of rAAV in liver of PKU mice were not reported for the treatments published by Mochiziki et al 27 or Oh et al 28 Serum hepatic and renal parameters (alanine aminotransferase ALT, aspartate aminotransferase AST, and gamma-glutamyltranspeptidase gGT), as well as inflammation parameters (interleukin-12 (IL-12) and tumor necrosis factor-a (TNF-a)) did not show any significant increase at 4 and at 20 weeks after portal or tail vein injections (not shown). This indicated that there was neither liver damage nor toxicity in rAAV vector-treated mice, which is also in agreement with reports from others.…”

“…26 Recently, two reports demonstrated the potential to correct PKU in a mouse model by AAV serotype 2-and 5-mediated gene transfer into liver. 27,28 However, route-independent, that is, tail-vein application, and long-term therapeutic effects in females were not achieved despite very high doses of rAAV vector administration (up to 10 14 viral particles per mouse). Since AAV serotype 2 and 5 have been shown to have a low efficiency in liver transduction, 12 alternative serotypes, as discussed above, might be employed for liverdirected gene transfer.…”

Potential of AAV vectors in the treatment of metabolic disease

“…Specific examples that have been investigated as therapeutic targets using rAAV include ornithine transcarbamylase (OTC) deficiency, 55 phenylketonuria (PKU) [56][57][58][59] homocytinuria, 60 methylmalonic aciduria (MMA) 61 and Canavan disease, 62,63 a cerebral organic aciduria.…”

“…Similar to results achieved for OTC deficiency, relatively robust phenotype correction has been achieved in mice, albeit more effective and durable in males, possibly as a consequence of higher initial levels of transduction. [56][57][58][59] The weakness of PKU as a target for gene therapy is the availability of effective dietary therapy.…”