Fig. 5: µDys gene transfer-mediated improvement of muscle resistance and partial rescue of DCG complex.

A Scheme of AAV9-µDys infection and doses used in DMD iPSC-derived MYOrganoids. B Evaluation of gene transfer efficiency by viral copy number (VCN) analysis. C µDys gene expression evaluated by RT-ddPCR represented as fold change to GAPDH gene expression. D Protein expression analysis by capillary western blot in isogenic iPSC (IsoCTR and DMDdEx8-9) derived MYOrganoids infected with low and high doses of µDys versus non-infected (–) IsoCTR (N = 3). E Percentage of drop force to calculate percentage of muscle force loss (Iso2-Iso1) after 10 eccentric contractions in isogenic cell lines, IsoCTR and DMD dEx8-9, and CTR1, CTR2 and DMD dEx45, DMDdEx8-43-derived MYOrganoids receiving low and high doses of µDys. N = 6 (F) Half decreasing time (Time ½ Fmax) in all CTR and DMD MYOrganoids treated or not with µDys at low and high dose. N = 5–8. G Histological evaluation of Dystrophin Glycoprotein Complex (DGC) upon infection, by immunostaining for Dystrophin (DYS), α-Dystroglycan (α-DG) and β- Dystroglycan (β-DG) in transversal sections of Isogenic CTR and DMD-iPSC-derived MYOrganoids. Scale bar: 40 µm and H relative quantification in all control and iPSC-derived MYOrganoids N = 4–5. Data are presented as means ± SEM. Statistical analysis was performed with an two-way analysis of variance test (for D) and ordinary one-way analysis of variance test (for all the other panels) with Šidák correction in multiple comparison tests(*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns = not significant) with multiple comparisons corrected with Dunnett’s test (F = 3,24 and degree of freedom = 24).