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. 2024 May 7;9(11):e165869. doi: 10.1172/jci.insight.165869

Figure 2. Adeno-associated virus-mediated striated muscle expression of micro-dystrophin constructs.

Figure 2

(A) Male D2.mdx mice were injected via tail vein with AAV carrying 1 of 4 CK8-driven MCDs (2x1014 gc/kg) at 1 month of age. (B) Microdystrophin gene therapy can result in premature death. Survival curve of D2.WT and D2.mdx untreated or treated with each of the 4 MDCs. None of the MDCs restored the lifespan of treated animals to that observed with DBA/2J WT animals (pair-wise Log-rank test; *P < 0.05 vs DBA/2J WT; Bonferroni correction). Two microdystrophin constructs (MDC1 and MDC4) led to premature death of treated mice (pair-wise Log-rank test; ◊P < 0.05 vs D2.mdx; Bonferroni correction). (C) Western blots of lysates of heart and gastrocnemius muscles from D2.mdx animals each transduced with 1 of the 4 MDCs. The 5-repeat MDCs examined (MDC2-4) show similar expression in both muscles, while the 4-repeat MDC1 was expressed at levels several-fold higher in comparison (~8- and ~6-fold higher in heart and gastrocnemius, respectively; n = 3–6, P < 0.001, 1-way ANOVA; ***P < 0.001 vs. mDC1, Tukey post hoc comparison). (D) Top 4 rows: Antibody-mediated labeling of heart transverse sections from D2.WT, D2.mdx untreated or treated with each of the 4 MDCs revealed sarcolemmal localization of microdystrophin proteins and restores sarcolemmal DGC. Bottom row: Antibody-mediated dystrophin labeling of gastrocnemius transverse sections demonstrated maintained sarcolemmal localization of each of the 4 MDCs that mirror sarcolemmal localization of endogenous dystrophin protein until the study endpoints. (E) Comparison of MDC4 expression vs endogenous full-length dystrophin in heart and gastrocnemius showed ~50- and ~5-fold overexpression, respectively (n = 4, Student’s t test, *P < 0.05, **P < 0.01). A majority of MDC4 was found to be associated with the membrane-enriched fraction of each tissue. Box-and-Whisker plots: minimum-to-maximum with 2nd and 3rd quartiles within the box, with a line that indicates the mean. Scale bars: 100 µm.