Figure 5.

Restoration of Dystrophin Expression after 2-Week Systemic Delivery of Z Polymer-Formulated PMO or Z-PMO Conjugate in mdx Mice Aged 4–5 Weeks

PMO (1 mg) only was used as controls. All other samples were from muscles treated with polymer-conjugated or simple formulated PMO (0.5 or 1 mg) in 100 μL saline. (A) Dystrophin was detected by immunohistochemistry with rabbit polyclonal antibody P7 against dystrophin. Blue nuclear staining with 4,6-diamidino-2-phenylindole (original magnification, ×200; scale bar, 200 μm). (B) The percentage of dystrophin-positive fibers in muscles treated with conjugated or simple formulated PMO. The numbers of dystrophin-positive fibers were counted in a single cross-section (n = 5, two-tailed Student’s t test, *p ≤ 0.05 compared with 1 mg PMO). (C) Detection of exon 23 skipping by RT-PCR. Total RNA of 100 ng from each sample was used for amplification of dystrophin mRNA from exon 20 to exon 26. The upper bands correspond to the normal mRNA, and the lower bands correspond to the truncated mRNA with exon E23 skipped. (D) Western blots demonstrate the expression of dystrophin protein from treated mdx mice in comparison with C57BL/6 and untreated mdx mice (50 μg of total protein was loaded for PMO/modified PMO and control mdx samples; 12.5 μg for the WT C57 control. A, TA; I, diaphragm; J, heart; Dys, dystrophin detected with monoclonal antibody Dys 1. α-Actin was used as the loading control.