Systemic antisense therapeutics inhibiting DUX4 expression ameliorates FSHD-like pathology in an FSHD mouse model

Figures

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  Figure 1. PMOs efficiently inhibit expression of DUX4 and downstream targets in immortalized FSHD myoblast cell cultures. Immortalized A5 myoblasts were differentiated for 2 days before the cells were treated with 10 μm PMOs through Endo-Porter–mediated transfection. Immortalized A10 or A5 cells receiving only Endo-Porter reagent were considered as untreated positive or negative control, respectively. Total RNA was extracted two days after PMO treatment. RT-qPCR quantification for DUX4 (A) and its targets: PRAMEF2 (B), TRIM43 (C), ZSCAN4 (D) and a marker of cell differentiation, MYH2 (E), are shown, relative to corresponding B2M expression. Cells in a parallel study were immunostained for all myosin isoforms using MF20 antibody. Cell fusion indexes were evaluated as the number of nuclei in MF20-positive myotubes containing ≥3 nuclei and expressed as the percentage of the total nuclei number in the image field (F). Representative cell images are displayed at magnification of ×100, scale bar = 100 μm, MF20 (green), DAPI (blue) (G). Statistical comparison (A–F) was by one-way ANOVA, followed by Tukey’s multiple comparisons test. Carets, asterisks or hashes indicate significances compared with…

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  Figure 2. In vivo experimental plan and effect of Vivo-PMO PACS4 treatment on body and muscle mass. Details of the experiment, including timepoints for functional tests, and changes in body weight of mice are presented (A). All mice received 2.5 mg/kg/biweekly tamoxifen (TMX) by intraperitoneal (IP) injection. MCM-D4 mice were further IP injected with 10 mg/kg/week of Vivo-PMO PACS4 (PACS4, n = 5), or 10 mg/kg/week of Vivo-PMO SCR (SCR, n = 4), considered as a negative control. HSA-MCM mice receiving volume-matched saline were considered as positive control (CTRL, n = 4). Animals were sacrificed 1 week after the last Vivo-PMO injection. Muscle mass of the gastrocnemius—GAS (B), quadriceps—QUAD (C) and tibialis anterior—TA (D) normalized to the corresponding body weight is displayed; muscles on both sides of the body were used. Data are shown as mean ± SEM. Statistical analysis was by two-way (A) or one-way ANOVA (B–D) followed by Tukey’s multiple comparisons test, P < 0.05 (*, ^), P < 0.01 (**, ^^), P < 0.001 (***), P < 0.0001 (****). In (A), black asterisks indicate significance between SCR and CTRL groups; gray asterisks or carets indicate significance between PACS4 and CTRL or PACS4 and…

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  Figure 3. Systemic antisense administration improves animal behavior and muscle function. Mouse open-field behavior was assessed every 3 days for three times using locomotor activity monitors. Data obtained from each mouse/time were averaged. Representative parameters of the animal behavior are shown in (A–C), while details of all parameters are presented in Supplementary Material, Table S2. Effects of antisense therapy were further evaluated via treadmill exhaustion tests 1 week prior to the first TMX injection (baseline) and then at weeks 2 and 4 post-injection. Total running time on treadmill was recorded and expressed as the time to fatigue as the percentage of baseline time (D). Following four weekly Vivo-PMO administration, mice were put under terminal anesthesia and in situ absolute force of both TAs/mouse was measured (E). Specific muscle force is displayed as a ratio of the absolute force and the TA cross-sectional area (F). Data are shown as mean ± S.E.M, n = 4–5 mice. Statistical analysis was by one-way ANOVA (A–D) or two-way ANOVA (E, F) followed by Tukey’s multiple comparisons test, P < 0.05 (*, ^), P < 0.01 (**, ^^), P < 0.001 (***), P < 0.0001 (****).

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  Figure 4. Vivo-PMO PACS4 treatment down-regulates expression of DUX4 and relevant genes. Following TMX-induced DUX4 expression and four weekly treatments with Vivo-PMOs, changes at mRNA levels in TA muscle were examined by RT-qPCR for DUX4 (A), Trim36 (B), Wfdc3 (C), Myh3 (D) and Pax7 (E), relative to corresponding Gapdh expression. Data are shown as means ± SEM; n = 4–5. Statistical comparison was by one-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****).

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  Figure 5. Effect of Vivo-PMO PACS4 therapy on muscle histopathology. Frozen TA muscle sections were stained for laminin, embryonic myosin heavy chain (eMyHC) and DAPI. The cross-sectional area (CSA) of the entire muscle section was automatically scored by MuscleJ (A). Laminin staining was used for identifying the fiber perimeter. An average of 3200 myofibers/TA was examined and displayed as the total number of myofibers (B) or the number of myofibers positive with eMyHC staining (C), per mm2 of the CSA. Fibrotic area in TA muscle was semi-automatically evaluated and expressed as percentage of the area positive for collagen of the muscle CSA (D). mRNA expression of gene indicative for fibrotic response, Col1α1 (E) and Tgfβ1 (F), was quantified by RT-qPCR. Statistical comparison was by one-way ANOVA followed by Tukey’s post hoc test (A–F). Data are shown as means ± SEM; n = 4–5; P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****). Representative images of the entire TA cross-sections co-stained with eMyHC (red), laminin (green), DAPI (blue) (G), or single stained with collagen VI (H) are shown at magnification of ×100, scale bar = 500 μm. Corresponding enlarged images at higher…

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  Figure 6. Vivo-PMO PACS4-mediated amelioration of muscle atrophy and myofiber type switching. Frozen TA muscle sections were immunostained for laminin (blue), MyHC IIA (green), MyHC IIB (red); unstained fibers were considered as MyHC IIX. The number of MyHC-positive fibers was automatically scored by MuscleJ and is expressed as the percentage of the total number of all myofibers within the entire muscle sections (A–C). Laminin staining was used for identifying the fiber sarcolemma and subsequent analysis of the minimal Feret’s diameter of myofibers. Histograms of frequency distribution (D–F) and the mean of the diameter of each myofiber type (G–I) are presented. Data are shown as mean ± SEM, n = 4–5. Statistical comparison was by one-way ANOVA and Tukey’s post hoc test (A–C, G–I); P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****). Representative images of the entire TA muscle sections are shown at magnification of ×100, scale bar = 500 μm; enlarged images at higher magnification are shown in the subsets, scale bar = 50 μm. Corresponding cartographic images created by MuscleJ software are also displayed, with MyHC IIA, IIB and IIX fibers color-coded as purple, light blue and…