Fig. 6: evoMmeFz2 and evoMmeFz2-HMG-D restore dystrophin expression in humanized DMD^{ΔmE5051, KIhE50/Y} mice following single AAV injection.

a Schematics of in vivo intramuscular (IM) injection of single AAV9-evoMmeFz2 or evoMmeFz2-HMG-D construct into the tibialis anterior (TA) muscle of the right leg of 3-week-old male DMD^{ΔmE5051, KIhE50/Y} mice. b Schematic representation of the exon-skipping strategy used by evoMmeFz2 or evoMmeFz2-HMG-D to restore the correct open reading frame (ORF) of the DMD transcript. c Gel electrophoresis was performed to analyze RT-PCR products from the muscle tissue of DMD^{ΔmE5051, KIhE50/Y} mice, and the experiment was repeated three times with similar results. Genomic (d) and RNA (e) indel editing events were analyzed by targeted-amplicon sequencing 3 weeks after intramuscular injection. f Immunofluorescence staining for DMD showed the restoration of dystrophin expression 3 weeks after TA injection of evoMmeFz2 or evoMmeFz2-HMG-D. The staining of dystrophin and spectrin proteins is depicted in green and purple, respectively. Scale bar: 100 μm. g Quantification of Dys⁺ fibers in cross sections of TA muscles. h Western blotting analysis was performed to assess dystrophin and vinculin expression in TA muscles 3 weeks after injection with AAV9-evoMmeFz2, AAV9-evoMmeFz2-HMG-D, or saline. Vinculin protein levels were used as an internal loading control. i The percentage of recovered dystrophin was quantified by grayscale intensity analysis. Data represent mean ± s.e.m. of three independent biological replicates. The p-value was determined using an unpaired two-tailed Student’s t test. Each dot represents an individual mouse for (d, e, g, and i). Source data are provided as a Source Data file.