Dose- and genotype-dependent cardiac arrhythmia and sudden death in rats following microdystrophin gene therapy

Figures

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  Figure 1. Follow-up on WT and DMDmdx rat body weights after injection with vehicle or GNT0004 product Data are shown as mean ± SEM. Mo, month.

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  Figure 2. hMD1 protein in WT and DMDmdx rats’ muscles 6 months after administration of vehicle or GNT0004 Analyses were performed on samples obtained from animals still alive at 6 months p.i. (i.e., n = 6–10 per experimental group). (A) Western blot analysis of hMD1 expression on total proteins extracted from biceps femoris, heart, and diaphragm of injected rats. Representative results are presented for one animal of each experimental group. Each blot was stained with MANEX-1011C to reveal the presence of the 427-kDa dystrophin protein (in WT rat) and the 138-kDa hMD1 protein. Anti-α-tubulin antibody was used as the loading control. (B) Quantification of hMD1 protein expression by Simple Western from total proteins extracted from biceps femoris, heart, and diaphragm of injected rats. Quantification was done respective to the level of total proteins and expressed as arbitrary units (a.u.). (C) Quantification of hMD1-positive muscle fibers after immunolabeling of biceps femoris and heart sections using the NCL-DYS3 antibody, which specifically recognizes the human dystrophin. (D) Immunohistochemical images of hMD1 expression in the biceps femoris and heart of injected rats. Representative…

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  Figure 3. Muscle histopathology and circulating biomarkers values measured 6 months after vehicle or GNT0004 administration Analyses were performed on samples obtained from animals still alive at 6 months p.i. (i.e., n = 6–10 per experimental group). (A) Semi-quantification of histopathological lesions evaluated in skeletal muscles (including diaphragm) and cardiac muscle. (B) Assessment of fibrosis in the heart. (C) Transverse heart sections stained with picrosirius red to visualize connective tissue (purple). Foci of ventricular fibrosis were sometimes observed (arrowhead). (D) Circulating creatine kinase (CK), aspartate aminotransferase (AST), aspartate aminotransferase (ALT), and lactate dehydrogenase (LDH) values. For (A), (B), and (D), individual and mean values (±SEM) are shown. Statistical analyses were performed using a nonparametric Kruskal-Wallis test followed by a post hoc Dunn’s multiple comparisons test (∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05).

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  Figure 4. Grip test assessment of skeletal muscle function and fatigue after vehicle or GNT0004 administration Analyses were performed at 3 (A) and 6 (B) months p.i. on animals still alive at the time of analysis (i.e., n = 7–10 per experimental group). Data plots show the mean forelimb grip force (g/g normalized to body weight [BW]) ± SEM collected over five successive grip test trials. Statistical analyses were performed using a nonparametric Kruskal-Wallis test followed by a post hoc Dunn’s multiple comparisons test (∗p ≤ 0.05 vs. corresponding WT group [treated with vehicle or GNT0004] and ¤p ≤ 0.05 vs. corresponding vehicle-treated group [WT or DMDmdx]). Cross-trial evolution of force was analyzed using the Friedman test followed by Dunn’s post-hoc test (§p ≤ 0.05 vs. trial 1 for the same group).

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  Figure 5. Assessment of hMD1 expression on cardiac function Analyses were performed on animals still alive at the time of analysis (i.e., n = 6–10 per experimental group). (A) Electrocardiographic evaluation at 6 months after vehicle or GNT0004 administration. Heart rate, PR interval, QT interval, and QTc interval (QT interval corrected with heart rate) were measured. (B) Echocardiography and pulsed Doppler evaluation at 6 months after vehicle or GNT0004 administration. For structural parameters, left ventricle (LV) anterior wall thickness and diameter were measured during diastole from long-axis images obtained by M-mode echocardiogram. For diastolic function, isovolumetric relaxation time (IVRT), deceleration time (DT), and E/A ratio were measured using pulsed Doppler with an apical 4-chamber orientation. For systolic function, ejection fraction was also measured during diastole from long-axis images obtained by M-mode echocardiogram. (C) Quantitative analyses of ECG results obtained using telemetry. The mean number of rhythm disorder events measured per session (i.e., each week) was evaluated for each animal. The results of each experimental group were compared, considering the total…

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  Figure 6. Quantification of endogenous rat dystrophin levels in WT and DMDmdx rat muscles 6 months after administration of vehicle or GNT0004 Analyses were performed on samples obtained from animals still alive at 6 months p.i. (i.e., n = 6–10 per experimental group). (A) Quantification of endogenous rat dystrophin protein expression levels by western blot from total proteins extracted from biceps femoris, heart, and diaphragm of injected rats. Each blot was stained with MANEX-1011C to detect the presence of the 427-kDa dystrophin protein. Quantification was done respective to the level of total proteins and expressed as arbitrary units (a.u.). (B) Quantification of hMD1-positive muscle fibers after immunolabeling in biceps femoris and heart sections using the NCL-DYS2 antibody, which specifically recognizes the endogenous rat dystrophin. The black arrow shows the differences observed in the heart samples between the different groups of WT animals. Individual and mean values (±SEM) are shown. Statistical analyses, performed using a nonparametric Kruskal-Wallis test followed by a post hoc Dunn’s multiple comparisons test, showed no significant differences between the different groups.

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  Figure 7. DAPC components in rat heart samples at 6 months after administration of vehicle or GNT0004 Analyses were performed on samples obtained from WT (A) and DMDmdx (B) rats that were still alive at 6 months p.i. (i.e., n = 6–10 per experimental group). Representative images of immunolabeling with antibodies against α-sarcoglycan (α-SG), rat native dystrophin (DYS2), hMD1 (DYS3), γ-sarcoglycan (γ-SG), and β-dystroglycan (β-DG). Compared to vehicle-injected WT rats, the WT rats injected with 4.2 × 1014 vg/kg of GNT0004 displayed subsarcolemmal clusters of α-SG (arrowhead) in fibers with low residual rat dystrophin expression but with high levels of hMD1 expression (open arrowhead). Fibers of interest were identified (°) in serial sections. Scale bar: 50 μm.