Figure 5: Analysis of cMD1 expression by immunostaining and western-blot in muscular biopsies of GRMD dogs injected with rAAV2/8-Spc512-cMD1 by the IV route.

(a–c) Dystrophin immunostaining (NCL-DYSB) on transverse sections of muscle samples. Representative results are presented for two treated GRMD dogs injected with different doses of the rAAV2/8-Spc5.12-cMD1 vector by the IV route: dog IV2, injected with 1 × 10¹⁴ vg kg⁻¹ (a) and dog IV7, injected with 2 × 10¹³ vg kg⁻¹ (b). A healthy (WT) dog is also presented as control (c). For the treated dogs, different muscle samples were obtained after surgical biopsies performed before injection, at 3.5 months post injection, at 8 months post injection and at 14 months post injection (only for Dog IV2 for this latter time point). The level of cMD1-positive fibres is indicated above each panel, as well as the number of vector genomes per diploid genomes detected by qPCR in the same muscle sample. Scale bar, 100 μm. (d) Western blot analysis of total proteins (50 μg) extracted from muscles samples. Representative results are presented for two other treated GRMD dogs, injected with different doses of the rAAV2/8-Spc5.12-cMD1 vector by the IV route: dog IV4, injected with 1 × 10¹⁴ vg kg⁻¹ and dog IV6, injected with 2 × 10¹³ vg kg⁻¹. Then, 50 μg of total proteins extracted from GRMD myoblasts transduced with the rAAV2/8-Spc5.12-cMD1 vector were used as positive control, as well as 25 to 75 μg of total proteins extracted from a skeletal muscle of a WT dog. The blot was stained with MANEX-1011C to reveal the presence of the 427 kDa dystrophin protein (WT dog) and the 138 kDa cMD1 protein, with an anti-GAPDH antibody as a loading control. The level of cMD1-positive fibres detected by immunostaining as well as the number of vector genomes per diploid genomes detected by qPCR from the same muscle samples are indicated under each panel.