Figure 1.

Simultaneous Liver and Muscle Targeting Improves Transgene Expression in Dystrophic Muscle

(A) Five-week-old Sgca^−/− mice were intramuscularly (i.m.) injected (tibialis anterior [TA]) at day (D) 0 with 5 × 10⁹ vg/mouse of AAV6-SPc5.12-hSGCA vector. Five days after intramuscular injection, mice received an intravenous injection (IV) with either 5 × 10¹¹ vg/mouse of AAV9-hAAT-hSGCA (Muscle-Liver group) or an empty AAV9 vector (Muscle group). In parallel, a group of mice injected intramuscularly with PBS and intravenously with the empty AAV9 vector was used as control (Control group). Two months after treatment, mice were sacrificed, and tissues were collected. (B) Hematoxylin phloxine saffron staining (HPS; upper panel; scale bar: 100 μm) performed in TA. (C) Immunostaining with anti-hSGCA (green), CD8 (red), and DAPI (blue) performed in TA. White arrows indicate CD8 cells. Scale bar: 50 μm. (D) miR-206 levels measured in TA and represented as fold-change versus wild-type C57BL/6J mice (dotted line in the graph). (E) Vector genome copy number (VGCN) per diploid genome measured in TA. (F) Anti-hSGCA IgG titers measured by ELISA using recombinant hSGCA protein. (G and H) CD8 (G) and IFN-γ (H) mRNA measured in TA. (I) IFN-γ secretion from recombinant hSGCA-stimulated splenocytes measured by ELISA. Data were expressed as mean ± SD. Statistical analyses were performed by Student’s t test in (D), (E), and (G)–(I); a non-parametric Mann-Whitney test was used for (F) (*p < 0.05, ^†p < 0.05 as indicated, n = 2 for Control group, n = 4 for Muscle group, and n = 6 for Muscle-Liver group).