227. Sustained Correction of Hyperphenylalaninemia in Female Pahenu2 Mouse by a Self-Complementary Adeno-Associated Virus Vector
Authors: Akihiro Kume, Tsuyoshi Ogura, Takashi Matsushita, Hiroaki Mizukami, Masashi Urabe, Takashi Okada, Keiya Ozawa
Journal: Molecular Therapy (2006)

The classic form of phenylketonuria (PKU) is caused by deficiency of phenylalanine hydroxylase (PAH), a critical liver enzyme converting phenylalanine (Phe) to tyrosine. If untreated, elevated serum Phe levels predispose afflicted children to severe brain damage. The present therapy for PKU mandates strict restriction of protein intake, preferably for the lifetime. Therefore long-term curing means such as gene therapy are awaited. Among preclinical studies with a mouse model of PKU (Pahenu2 strain carrying a missense mutation in the PAH gene), vectors derived from adeno- associated virus (AAV) have given the most promising results. We and other investigators have successfully corrected hyperphenylalaninemia by portal delivery of recombinant AAV vectors. Similar to other AAV-mediated liver transduction models, however, female animals required much higher vector dose than males, and tended to lose the therapeutic effect over time. Aiming at better gene transfer to the liver, we investigated the efficacy of the self-complementary AAV system in Pahenu2. A self-complementary AAV vector (scAAV8/LP1-mPAH) was constructed by replacing the human factor IX gene of scAAV8/LP1-hFIX (a gift from Dr. JT Gray, St. Jude Research Hospital) with the murine PAH gene. For comparison, a conventional single strand AAV8 vector (AAV8/CAG-mPAH) was constructed by transferring the PAH expression cassette from AAV5/CAG-mPAH (Mochizuki S et al, Gene Therapy 11:1081, 2004). AAV type 8 capsid was chosen for packaging because of its stronger liver tropism than AAV2 and AAV5. In most cases, vectors were given to the peritoneal cavity of Pahenu2 mice of 4-5 weeks of age, because AAV8 vectors accumulate mainly in the liver regardless of the route of administration. In parallel to our previous experiment with AAV5, female PKU mice were refractory to AAV8/CAG-mPAH. While 1x1012 particles of the vector reduced blood Phe to the therapeutic level in males, no Phe reduction was obtained by injecting tenfold more AAV8/CAG-mPAH (1x1013 particles) to females. Portal injection of the same vector dose (1x1013 particles of AAV8/CAG-mPAH) was effective initially, but hyperphenylalaninemia revisited after 16 weeks of the treatment. In contrast, the self-complementary AAV vector brought about a dramatic effect. Within 2 weeks after receiving 3x1012 or 1x1013 particles of scAAV8/LP1-mPAH, blood Phe was completely normalized in the female Pahenu2 mice. The therapeutic effect has been stable up to 16 weeks (3x1012) and 30 weeks (1x1013), and the follow-up study is ongoing. Injection of 1x1012 particles of scAAV8/ LP1-mPAH had no effect, suggesting that the threshold dose lies somewhere between 1x1012 and 3x1012 particles when the vector is administered intraperitoneally. These results indicated that the efficacy of scAAV8/LP1-mPAH was about tenfold higher than AAV8/CAG-mPAH. Dissection of the mechanisms for this superior performance of scAAV8/LP1-mPAH should help further vector improvement for PKU treatment.