343. Characterization of Delivery Routes of AAV Vectors for Neonatal Gene Transfer
Authors: Tsuyoshi Ogura, HIroaki Mizukami, Jun Mimuro, Takashi Okada, Hiromi Hamada, Akihiro Kume, Hiroyuki Yoshikawa, Yoichi Sakata, Keiya Ozawa
Journal: Molecular Therapy (2004)

Gene transfer into a fetus or neonate is considered to be a fundamental approach to the treatment of genetic diseases, which cause irreversible manifestations in adulthood, and is potentially advantageous in transducing tissues with relatively low vector doses. Because of its safety and long-term transgene expression, adeno-associated virus (AAV) vector is one of the promising vehicles for this purpose. Although the potential utility has been suggested, systematic comparison of administration routes of AAV vectors has not been fully investigated, especially for neonates. In this study, we explored the optimal delivery route of AAV vectors for neonatal gene therapy in mice. For this purpose, vectors carrying human coagulation factor IX (hFIX) gene under cytomegalovirus (CMV)-derived promoter using either AAV1 or AAV5 capsids were administered into immune-competent neonatal mice (C57BL/6) within 24 hours after birth. As for delivery routes, injections into muscles of lower limbs, a cervical vein, and a peritoneal cavity were tested. Based on our previous observations, AAV1-CMV-hFIX vectors were used for intramuscular injection and AAV5-CMV-hFIX vectors for intravenous and intraperitoneal cavity injection. Each neonate mouse received a dose of 1 × 10(10) genome copy per body. Plasma concentrations of hFIX were periodically monitored after the vector injection. As a result, intramuscular injection showed the highest hFIX concentration (142.0 ± 67.5 ng/ml), followed by intraperitoneal cavity injection (21.8 ± 5.8 ng/ml) and intravenous injections (10.7 ± 1.5 ng/ml). Dose escalation study for intramuscular injections (5 × 10(10) genome copy of AAV1-CMV-hFIX) showed a higher hFIX concentration (243.2 ± 72.6 ng/ml), a level that is considered within therapeutic range, and the expression lasted for more than 24 weeks. Additionally, we examined the difference of transduction efficiency by sex, because liver-targeted transduction with AAV vectors is known to be inefficient for adult female mice. To validate the sex-related difference in gene expression in neonate and adult mice, each mouse was injected with 5 × 10(10) genome copy of AAV1-CMV-hFIX vectors intramuscularly or 3 × 10(11) genome copy of AAV5-CMV-hFIX vectors into peritoneal cavity. AAV5-CMV-LacZ vectors were also used for evaluating gene expression throughout the body. Our study revealed that the sex-related difference in transgene expression was observed for intraperitoneal cavity injection into adult mice but not into neonate mice 10 weeks following injection (male vs. female: 98.3 ± 24.8 vs. 10.8 ± 3.8 ng/ml in adult, 214.4 ± 42.6 vs. 178.2 ± 38.9 ng/ml in neonate, respectively). Interestingly, X-gal-staining after intraperitoneal cavity injection of AAV5-CMV-LacZ vectors showed that the sex-related difference in transgene expression was apparent in adult peritoneal epithelium, and that such a difference was not observed in groups with neonatal injection. These findings would be valuable for designing strategies of neonatal gene therapy in terms of delivery routes of AAV vectors.