900. Overcoming Gender-Specific Barrier of AAV-Mediated Gene Therapy by Neonatal Intervention
Authors: Akihiro Kume, Tsuyoshi Ogura, Hiroaki Mizukami, Shigeo Kure, Takashi Okada, Masashi Urabe, Takashi Matsushita, Keiya Ozawa
Journal: Molecular Therapy (2005)

Phenylketonuria (PKU) is one of the common genetic human disorders, mostly resulting from deficiency of a liver enzyme phenylalanine hydroxylase (PAH). Without a strict and continuous diet therapy starting at early infancy, accumulated phenylalanine (Phe) in the body leads to severe mental defect. A mouse model of PKU (Pah enu2) was created by chemical mutagenesis, and affiliated animals show a closely resembled phenotype to human PKU patients with elevated blood Phe, cognitive deficits and hypopigmentation. We have successfully treated male Pah enu2 mice by liver-directed gene therapy with an adeno-associated virus (AAV) type 5 vector (AAV5/CAG-mPAH). The vector dose of 1 |[times]| 10 13 vector genomes (vg) was partially effective, and the blood Phe level was completely normalized with higher doses. The long-term correction of hyperphenylalaninemia was associated with normalized basal and exploring activity, indicating an improvement in brain function (Mochizuki S et al, Gene Therapy 11:1081, 2004). Conversely, 1 |[times]| 1014 vg of the vector was required to correct hyperphenylalaninemia in female Pahenu2 mice, and the therapeutic effect was transient. Other investigators have documented similar refractoriness of female liver to AAV. Aiming at a better transduction of the female Pahenu2 mouse liver, we investigated the efficacy of an AAV type 8 vector with the same expression cassette (AAV8/CAG-mPAH). First, we injected 1 |[times]| 1013 vg of AAV8/CAG-mPAH into the portal vein of adult female Pahenu2 mice. The blood Phe level in the treated mice was completely normalized within 2 weeks. The efficacy was equal to or greater than 1 |[times]| 1014 vg of AAV5/CAG-mPAH, suggesting that AAV8 was tenfold potent than AAV5. However, the therapeutic effect was transient as was observed in our previous experiments with AAV5. After 16 weeks of AAV8 injection, the blood Phe level was gradually increased, and returned to the pretreatment level by week 24. We also monitored the in vivo PAH activity by injecting 1-13C-Phe. This noninvasive assay confirmed that the metabolic activity waned over time. Next, we addressed whether earlier intervention to Pahenu2 mice could overcome the poor transduction of female liver. We observed that AAV8 accumulated in the liver following intravenous injection into neonates and the reporter genes well expressed in both males and females (see accompanied abstracts by Ogura T et al). Based on this observation, 5 |[times]| 1011 vg of AAV8/CAG-mPAH was administered to Pahenu2 mice via jugular vein on day 1. Four weeks later, the blood Phe level was significantly lower than the age-matched Pahenu2 controls (treated males 22.1 |[plusmn]| 1.9mg/dl, treated females 21.0 |[plusmn]| 0.8 mg/dl vs. untreated 32.4 |[plusmn]| 2.7 mg/dl; P < 0.001). Coat color of the treated mice was slightly darker than the untreated animals, supporting a marginal effect by the intravenous AAV8/CAG-mPAH. These results suggest that neonatal gene transfer may overcome the gender-specific refractoriness to AAV vectors.