Micro-dystrophin gene therapy demonstrates long-term cardiac efficacy in a severe Duchenne muscular dystrophy model

Figures

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  Figure 1. AAV6-μDys5 treatment prevents whole-heart dysfunction and myocardial strain abnormalities in Fiona/dko mice (A–D) Longitudinal echocardiography at 6, 9, 12, 15, and 18 months of age in Fiona/dko and μDys5-treated Fiona/dko mice, showing (A) ejection fraction (EF), (B) fractional shortening (FS), (C) interventricular septal width, end systole (IVS,s), and (D) left ventricular internal diameter, end systole (LVID,s). Fiona/dko mice had significantly reduced function and structural remodeling at 6 and 9 months of age, which were prevented by treatment with AAV6-μDys5 at 4 weeks of age. Fiona/dko mouse heart function then improves beginning at 12 months of age through the 18 month endpoint. Heart rates were maintained at an average of 450 bpm throughout echocardiography. Welch’s t test was performed for each parameter to compare groups at each time point (lines under x axes). Values are expressed as mean (markers) ± SEM (error bars). ∗p ≤ 0.05, ∗∗p ≤ 0.01. For (A)–(D): n = 4 males/5 females for Fiona/dko from 6 to 12 months, then n = 3 males/5 females from 15 to 18 months; n = 4 males/3 females for μDys5 group at all timepoints. (E–G) Circumferential strain rate analysis showed a…

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  Figure 2. μDys5 protein is expressed and uniformly localized at cardiomyocyte membranes throughout Fiona/dko hearts at 18 months (A) Western blot analysis of heart lysates from 3-, 12-, and 18-month-old Fiona/dko, 18-month-old μDys5 treated Fiona/dko, and C57BL/10 (C57) wild-type control mice. Blots were incubated with antibodies to detect both mouse and human utrophin (MANCHO3) (top) and dystrophin (MANDYS1) (bottom). Ponceau S staining shows equivalent loading between samples. Utrophin (MANCHO3) blot (top) shows that neither mouse nor human utrophin is present in any of the untreated or treated Fiona/dko hearts, validating complete knockout of mouse utrophin and that the “Fiona” human utrophin transgene does not become expressed in heart through 18 months of age. Dystrophin (MANDYS1) blot (bottom) shows that full-length dystrophin is present in C57 hearts and that micro-dystrophin is present only the μDys5-treated Fiona/dko mice. (B) Representative composites and zoomed immunofluorescence images of heart ventricular sections from treated and untreated Fiona/dko mice stained with a polyclonal antibody that detects the amino terminus of both mouse and human dystrophin (red). Zoomed panels…

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  Figure 3. AAV6-μDys5 treatment prevents cardiac fibrosis in Fiona/dko mice (A) Representative composite images of hematoxylin and eosin-stained transverse ventricular heart sections showing the complete prevention of histopathology in 18-month-old AAV-μDys5 (μDys5)-treated compared with untreated Fiona/dko mice. (B) Representative composite images of fibronectin immunofluorescence-stained (red) heart sections demonstrate absence of replacement fibrosis in μDys5 compared with Fiona/dko mice at 18 months of age. Zoomed images show areas within white boxes in composites of fibronectin (red) or co-staining with laminin (green) and merged images demonstrating fibrotic replacement in myocardium. Images shown are from samples near the mean for each group quantified in (C). (C) Quantification of fibrosis, shown as the percentage of area of ventricular composite sections containing fibronectin staining, demonstrates that μDys5 prevents fibrosis through 18 months of age compared with Fiona/dko hearts. Only small amounts of fibronectin normally surrounding vessels contributed to the quantification in μDys5 hearts. Statistical analysis performed with unpaired t test with Welch’s correction; ∗∗∗∗p <…

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  Figure 4. AAV6-μDys5 treatment prevents tenascin C and collagen accumulation, but collagen packing changes with age in Fiona/dko mice (A) Representative composite images of tenascin C immunofluorescence-stained heart sections demonstrate prevention of tenascin localization in μDys5 compared with Fiona/dko mice at 18 months of age and no change with age between 12- and 18-month-old Fiona/dko hearts. Images shown are from samples near the mean for each group quantified in (C). (B) Polarized images of Picrosirius red-stained heart sections show a difference in collagen packing in 12- compared with 18-month-old untreated and treated Fiona/dko mice as well as an overall decrease in collagen amount in μDys5-treated compared with untreated Fiona/dko mice at 18 months. Images shown are from samples near the mean for each group quantified in (D). (C) Quantification of tenascin C shown as the percentage of area of ventricular composite sections containing staining (red) demonstrates that μDys5 prevents increased tenascin through 18 months of age compared with 12- and 18-month-old Fiona/dko hearts. Tenascin percentages were not significantly different between 12 and 18 months of age for untreated…

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  Figure 5. AAV6-μDys5 treatment prevents cardiac inflammation in Fiona/dko mice (A) Representative composite (top) and zoomed (bottom) images of CD45 immunohistochemical staining (brown) of leukocytes in heart sections of 18-month-old untreated and μDys5-treated Fiona/dko mice. Zoomed images show areas within black boxes in composites. Images shown are from samples near the mean for each group quantified in (B) (untreated, F; μDys5 treated, M). (B) Quantification of inflammation shown as the percentage of area of ventricular composite sections containing CD45 pan-leukocyte staining demonstrates that μDys5 prevents inflammatory infiltrate through 18 months of age compared with untreated Fiona/dko hearts. Dot plots display total n analyzed for each group. Statistical analysis performed with unpaired t test with Welch’s correction (p = 0.0003). ∗∗∗p ≤ 0.01. Scale bars: 100 μm.