Analysis of hepatocyte targeting with exo-AAV vectors. Male C57BL/6 mice (n = 5) injected IV with 109 vg per mouse of exo- or standard AAV8- hF.IX vectors. (A) Plasma levels of hF.IX measured by ELISA 14 days posttreatment. (B) VGCN per diploid genome in parenchymal and nonparenchymal cells isolated 14 days posttreatment. (C) Immunostaining for hF.IX in livers from mice treated with standard (top) or exo-AAV8 (middle) vectors, or phosphate-buffered saline (PBS; bottom) performed 14 days posttreatment. hF.IX is shown in red and nuclear staining (Dapi) in blue. Representative sections were acquired at a magnification of ×20 (insets at ×300). (D) hF.IX quantification after staining in liver. Analysis was performed using the Cellprofiler software and the percentage of positive hepatocytes was determined based on high (200-5000 arbitrary units), or medium (130-199 arbitrary units) signal intensity. Background signal (1-129 arbitrary units) was not calculated. Data are shown as mean ± SEM (for details of analysis, see supplemental Methods). For hF.IX plasma levels and VGCN, statistical significance was determined with the Mann-Whitney U test; for signal intensity, a 1-way ANOVA was used to determine statistical significance; for VGCN, an unpaired Student t test was used. *P < .05; **P < .05.
