6. AAV8-Mediated Transgene Expression in Mice and Non-Human Primates
Authors: Hiroaki Mizukami, Jun Mimuro, Akira Ishiwata, Fumiko Ono, Takashi Matsushita, Takashi Okada, Masashi Urabe, Akihiro Kume, Keiji Terao, Yoichi Sakata, Keiya Ozawa
Journal: Molecular Therapy (2006)

Vectors using AAV8 capsid have shown remarkable results in liver-directed gene transfer in mice. However, the utility of AAV8 vectors in larger animal models have scarcely been described. Here we report our results with mice and non-human primates (cynomolgus macaque) to test the usefulness of AAV8 vectors for human applications. As a transgene, we chose macaque coagulation factor IX (FIX) with a mutation at position 262 (macFIXT262A). Based on our previous study, this molecule carries minimal alteration and can be detected with a monoclonal antibody against human FIX, and an assay system utilizing this antibody has been established to quantitate macFIXT262A even in the presence of macaque FIX (J of Thromb and Haemost 2: 275-80, 2003). A stock of AAV8 vector encoding macFIXT262A driven by human alpha1-antitrypsin (hAAT) promoter with a liver specific enhancer was prepared. When the vector was injected into C57BL/6 mice intraportally at 1 × 1010 vg/body, plasma concentration of the transgene showed more than 100 % of the normal level. When the same vector was injected into a young adult male macaque at a dose of 1 × 1012 vg/kg, plasma concentration of the transgene was detectable throughout the observation period, but not recognizable as therapeutic level (< 0.1%). The efficacy was again tested in another male at a higher titer of 1 × 1013 vg/kg, and resulted in a similar outcome with a slightly higher level. Macaques were extensively immunosuppressed with FK506 and cyclophosphamide until 8 weeks after injection. To better understand these results, potential factors affecting transgene expression were analyzed. Neutralizing antibody against AAV8 capsid was not detectable before injection. Antibody against transgene product was not recognizable. At present, none of the factors inhibiting transgene expression is identified, implying a species-specificity of the efficacy of AAV8 vectors. Vectors using AAV8 capsid have shown remarkable results in liver-directed gene transfer in mice. However, the utility of AAV8 vectors in larger animal models have scarcely been described. Here we report our results with mice and non-human primates (cynomolgus macaque) to test the usefulness of AAV8 vectors for human applications. As a transgene, we chose macaque coagulation factor IX (FIX) with a mutation at position 262 (macFIXT262A). Based on our previous study, this molecule carries minimal alteration and can be detected with a monoclonal antibody against human FIX, and an assay system utilizing this antibody has been established to quantitate macFIXT262A even in the presence of macaque FIX (J of Thromb and Haemost 2: 275-80, 2003). A stock of AAV8 vector encoding macFIXT262A driven by human alpha1-antitrypsin (hAAT) promoter with a liver specific enhancer was prepared. When the vector was injected into C57BL/6 mice intraportally at 1 × 1010 vg/body, plasma concentration of the transgene showed more than 100 % of the normal level. When the same vector was injected into a young adult male macaque at a dose of 1 × 1012 vg/kg, plasma concentration of the transgene was detectable throughout the observation period, but not recognizable as therapeutic level (< 0.1%). The efficacy was again tested in another male at a higher titer of 1 × 1013 vg/kg, and resulted in a similar outcome with a slightly higher level. Macaques were extensively immunosuppressed with FK506 and cyclophosphamide until 8 weeks after injection. To better understand these results, potential factors affecting transgene expression were analyzed. Neutralizing antibody against AAV8 capsid was not detectable before injection. Antibody against transgene product was not recognizable. At present, none of the factors inhibiting transgene expression is identified, implying a species-specificity of the efficacy of AAV8 vectors.