Figure 2.

Dose-escalation study using intramuscular delivery of AAV1-U7E6E8 in golden retriever muscular dystrophy dog (GRMD). (a) Dystrophin immunostaining (NCL-DYS2 monoclonal antibody) on transverse sections of GRMD biopsies, 2 months after intramuscular injections of the tandem AAV1-U7E6E8 vector into the triceps brachii muscle. Dose-escalation: phosphate-buffered saline (PBS); 5 × 10¹⁰; 10¹¹; 2.5 × 10¹¹; 5 × 10¹¹; 10¹²; 2 × 10¹²; and 3.5 × 10¹² vg (A–H, respectively). (b) Quantification of the adeno-associated virus (AAV) genome copy number in samples of the dose-escalation study. (A–H) correspond to: PBS; 5 × 10¹⁰; 10¹¹; 2.5 × 10¹¹; 5 × 10¹¹; 10¹²; 2 × 10¹²; and 3.5 × 10¹² vg, respectively. (c) Proportion of dystrophin positive fibers in muscle samples following the dose-escalation study. For each condition, results are expressed as the mean of the percentage of dystrophin positive fibers obtained in five regions (±SD). (d) Detection of in-frame multi-skipped dystrophin mRNAs by nested reverse transcription-PCR (RT-PCR) from exon 3 to 10 in dose-escalation samples. (lane A: PBS) PCR products (792- and 663-bp fragments) correspond to out-of-frame mRNA variants lacking either exon 7 or exons 7 and 9, respectively. The RT-PCR pattern is changed accordingly to the AAV1-U7E6E8 dose. Two main fragments of 437-bp and 308-bp corresponding to in frame mRNAs lacking either exons 6 to 8 or 6 to 9 (white stars), respectively, are clearly detected at 10¹¹ vg and above. Additional bands on top of the in frame fragments correspond to classical hetero-duplex artifacts obtained during the course of the PCR amplification. (e) Western blot of total protein (100 µg) extracted from injected GRMD muscle samples stained with NCL-DYS2. Arrow indicates the full-length dystrophin as detected in samples from normal muscle used for comparison (25, 12, 6, and 3%—diluted in GRMD to keep up total protein content). Samples were also hybridized with an actin-antibody to validate protein loadings.