AAV8-driven liver-directed gene transfer is significantly delayed in hemophilia A mice. (A) Schematic showing delivery of AAV8–TBG-GFP to hemophilia A (F8^TKO) or control (unaffected littermate heterozygous) mouse. (B) Western blot analysis of GFP in control and F8^TKO mice after 15- and 30-days post–AAV8-GFP administration (IP). (C) Immunofluorescence of GFP staining in control and F8^TKO mice 15 days post–AAV8-GFP injection (IP). The dotted regions are zoomed in as inset. (D) ELISA assay of total liver GFP amount in control and F8^TKO at 15 days post–AAV8-GFP injection (P = .04). (E) Immunofluorescence of GFP staining in control and F8^TKO mice 30 days post–AAV8-GFP injection (IP). The dotted regions are zoomed in as inset. (F) ELISA assay of total liver GFP amount in control and F8^TKO 30 days post–AAV8-GFP injection (P = .06). (G) Immunofluorescence of GFP staining in control and F8^TKO mice 60 days post–AAV8-GFP injection (IV). The dotted regions are zoomed in as inset. (H) ELISA assay of total liver GFP amount in control and F8^TKO at 60 days post–AAV8-GFP injection (P = .07). (I) Immunofluorescence for caspase-3 showed an increased accumulation in F8^TKO mouse liver 15- and 30-days post–AAV8-GFP administration, which was not seen in matched control liver. All control mice used were unaffected littermate heterozygoes mice.