105. A Single Administration of Low Dosage (5 x1010 vp/kg) hFX/HDAd Complex Achieves Saturated hFVIII Levels in the Blood of Mice
Authors: Masataka Suzuki, Michele Martinez, Alice Tarantal, Brendan Lee
Journal: Molecular Therapy (2015)

The development of helper-dependent adenoviral vectors (HDAds) devoid of all functional viral genes has largely overcome cellmediated host immune responses associated with Ad viral proteins. However, a dose-related acute toxicity associated with systemic delivery has been a major obstacle to the clinical translation of Adbased vectors. Since HDAd has a large transgene cargo (up to 34 kb), we recently succeeded in producing an HDAd encoding two codon-optimized hFVIII variant N6 expression cassettes (HDAd2xcoN6). We evaluated whether HDAd2xcoN6 improves hFVIII expression and activity in vivo. Hemophilia A (HA) mice were injected with a sub-lethal (human) dose (5 x1011 vp/kg) of HDAd2xcoN6 or HDAd1xcoN6 encoding a single expression cassette. HA mice injected with HDAd2xcoN6 exhibited over 10-fold higher hFVIII expression and activity compared to those injected with an equal titer of HDAd1xcoN6. Thus, we evaluated hFVIII expression after a systemic administration of 5 x1010 vp/kg (one log-lower than a human sub-lethal dose) of HDAd2xcoN6 in non-human primates. One primate showed 2% hFVIII levels in plasma compared to pretreatment. Though that expression was transient, it represents the first time that an Ad-based vector has shown a detectable level of therapeutic gene expression in the blood after systemic administration with such a low dosage in a large animal model. However, this result also suggests that the improvement of transduction of HDAd and transgene expression in the liver is required for long-lasting, high hFVIII expression in a large animal model. Previous reports have revealed that the adenoviral capsid interacts with coagulation factor X (FX) in the blood after systemic administration, and the interaction leads to transduction of this complex into the liver in conjunction with highly expressing HSPGs (receptor of FX/Ad5 complex). The complex of Ad5 with human FX (hFX/HDAd) has a greater affinity to HSPGs than mouse FX does. We therefore evaluated forming a complex of HDAd with hFX in order to improve HDAd liver transduction. Mice injected with 5 x1010 vp/kg of hFX/ HDAd2xcoN6 complex sustained 100% hFVIII levels in the blood until 4 weeks after injection. In contrast, mice injected with same dosage of HDAd2xcoN6 alone had undetectable levels of hFVIII in the blood 4 weeks post-injection, similar to our observations in non-human primate. Although mice injected with the hFX/HDAd complex showed 10-fold higher transgene products in vivo compared to mice treated with HDAd alone, HDAd vector copies in the livers of mice injected with the hFX/HDAd complex were only 2-fold higher. Interestingly, mice injected with the hFX/HDAd complex exhibited 10-fold higher transgene expression at mRNA level in the liver compared to mice injected with HDAd alone. These results suggest that the hFX/HDAd complex can lead not only to high liver transduction, but also to improving transgene expression at the transcriptional level after systemic administration. We are now investigating how the hFX/HDAd complex improves transgene expression at the transcriptional level.