Figure 6
Quantitative analysis of dystrophin expression following a 6 week, repeat intramuscular injection regimen of NG-PEI2K(PEG550) 10 polyplexes. Tissues were analyzed following 10 twice-weekly intramuscular injections of either 1 or 5 μg of ESO complexed with NG-PEI2K(PEG550)10 copolymers, and harvested 6 wks after the first injection. (A) Dystrophin immunolabeling (Hoechst dye counterstained) and H&E staining of serial transverse sections from TA muscles from the 10 μg (1 μg × 10 injections) and 50 μg (5 μg × 10 injections) groups. (B) Number of dystrophin-positive fibers, obtained from whole transverse sections immunolabeled for dystrophin, was significantly lower in muscles injected with 10 μg ESO compared with the 50 μg group (P < 0.001, N = 4 muscles per group). (C) Western blots showing dystrophin expression in thick (60 μm) transverse cryosections taken adjacent to segments used for the fiber counts in panel B. Images show blots of dystrophin (top) and vinculin (bottom) obtained from the same gel. All samples contained 25 μg of total protein, and dystrophin expression as a percent of normal is indicated in parentheses below each lane. (D) Dystrophin expression determined from Western blots reached 20% of normal levels in muscles injected with NG-PEI2K(PEG550)10-ESO containing 50 μg ESO, which was significantly greater than the 10 μg group (P < 0.001, N = 4 muscles per group).