Classical phenylketonuria (PKU) is a metabolic disorder caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). If untreated, accumulation of phenylalanine will damage the developing brain of affected individuals, leading to severe mental retardation. Here, we show that a liver-directed PAH gene transfer brought about long-term correction of hyperphenylalaninemia and behavioral improvement in a mouse model of PKU. A recombinant adeno-associated virus (AAV) vector carrying the murine PAH cDNA was constructed and administered to PAHdeficient mice (strain PAH enu2 ) via the portal vein. Within 2 weeks of treatment, the hyperphenylalaninemic phenotype improved and completely normalized in the animals treated with higher vector doses. The therapeutic effect persisted for 40 weeks in male mice, while serum phenylalanine concentrations in female animals gradually returned to pretreatment levels. Notably, this long-term correction of hyperphenylalaninemia was associated with a reversal of hypoactivity observed in PAH enu2 mice. While locomotory activity over 24 h and exploratory behavior were significantly decreased in untreated PAH enu2 mice compared with the age-matched controls, these indices were completely normalized in 12-month-old male PKU mice with lowered serum phenylalanine. These results demonstrate that AAV-mediated liver transduction ameliorated the PKU phenotype, including central nervous system dysfunctions.
Background Classical phenylketonuria (PKU) arises from a deficiency of phenylalanine hydroxylase (PAH) that catalyses phenylalanine oxidation in the liver. Lack of PAH activity causes massive hyperphenylalaninemia and consequently severe brain damage. Preclinical studies showed that conventional adeno-associated virus (AAV) vectors could correct hyperphenylalaninemia in a mouse model of PKU, although limitations such as very large dose requirement and relative inefficiency in female animals were recognized.
Adenovirus and adeno-associated virus (AAV) vectors are increasingly used for gene transduction experiments. However, to produce a sufficient amount of these vectors for in vivo experiments requires large-capacity tissue culture facilities, which may not be practical in limited laboratory space. We describe here a large-scale method to produce adenovirus and AAV vectors with an active gassing system that uses large culture vessels to process labor- and cost-effective infection or transfection in a closed system. Development of this system was based on the infection or transfection of 293 cells on a large scale, using a large culture vessel with a surface area of 6320 cm2. A minipump was connected to the gas inlet of the large vessel, which was placed inside the incubator, so that the incubator atmosphere was circulated through the vessel. When active gassing was employed, the productivity of the adenovirus and AAV vectors significantly increased. This vector production system was achieved by improved CO2 and air exchange and maintenance of pH in the culture medium. Viral production with active gassing is particularly promising, as it can be used with existing incubators and the large culture vessel can readily be converted for use with the active gassing system.
Traditionally, skeletal muscle and liver are the preferred target organs for gene transfer to supply a transgene product into the systemic circulation. In this respect, adipose tissue presents a number of attractive features. However, adipose tissue transduction in vivo has not been feasible by conventional methods. To solve this issue, we tested the utility of excipients in adeno-associated virus (AAV) vector-mediated gene transfer and found that Pluronics are suitable for this purpose. In a histological analysis of adipose tissue in db/db mice, Pluronic F88 showed the greatest augmentative effect on beta-galactosidase expression in combination with the AAV1 vector. When the vector encoding mouse erythropoietin (Epo) was used in the same manner, increased plasma Epo concentrations were observed (230 +/- 80 versus 58 +/- 14 mU/ml). Moreover, the plasma Epo concentration returned to the normal level after the surgical removal of transduced adipose tissue. No damage was observed in the transduced tissue. Our results indicate that the proposed method is safe and efficient for gene transfer into adipose tissues, thus providing an alternative for supplemental gene therapy.
AAV vector administration to neonates using the i.p. route was clearly advantageous in obtaining robust transgene expression. Vector genomes and transgene expression were observed mainly in the peritoneal tissue. These findings indicate the advantages of neonatal gene therapy and would help in designing strategies for gene therapy using AAV vectors.
If ACC with fetal ventriculomegaly is associated with other malformations, a poor outcome is highly likely. Although the outcome of isolated ACC with fetal ventriculomegaly was generally better, >50 % of the patients had mild or moderate neurodevelopmental disabilities. These findings provide useful information for parental counseling.
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