AAV-mediated delivery of cFVII zymogen in FVII-G96E dogs results in stable transgene expression. (A) A PT clotting assay was used to indicate biological activity of the expressed cFVII transgene, following AAV administration (day 0, black arrow) in FVII-G96E dogs. The symbol for the AAV vector dose for each of the FVII-G96E dogs is shown. The first antigen level quantification after AAV administration was within 8 to 13 days. The time to reach the plateau of expression following AAV administration was as follows: N24, day 28; N25, day 50; N22, day 42; and Otis, day 87. (B) AAV administration (day 0, black arrow) in FVII-G96E dogs resulted in cFVII protein expression, determined by ELISA. The symbol for the AAV vector dose for each of the FVII-G96E dogs is shown. (C) Graphical representation of the relationship of AAV vector dose administered and the cFVII antigen levels (% normal) at the expression plateau in the FVII-G96E dogs. Values are shown as mean ± 1 standard deviation, obtained from the individual cFVII antigen levels at each time point after expression reached a plateau for each dog. The dotted line indicates the percent normal FVII that, when achieved, improves hemostasis in FVII-deficient patients. (D) Detection of cFVII transgene expression in N24 plasma (day 84) by fluorescence-based western blot (shown in gray scale). For comparison, recombinant WT cFVII (black arrow) was added in FVII-G96E canine plasma at the indicated concentration. All plasma samples were diluted 1:32 in PBS.