DG9 boosts PMO nuclear uptake and exon skipping to restore dystrophic muscle and cardiac function

Figures

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  Fig. 1. Cellular internalization of PMOs after DG9 conjugation. a Representative immunofluorescence images of del45 myoblasts, myotubes, and healthy cardiomyocytes after incubating with 3’-carboxyfluorescein-labeled DG9-PMO at 1 µM (green) for 24 h. Cell nuclei were labeled with DAPI. Scale 20 µm. b Time course of the uptake and nuclear localization of DG9-PMO (1 µM) in del45 myoblasts, myotubes, and healthy cardiomyocytes. The shaded region indicates the variations in intracellular DG9-PMO levels following its removal (n = 3). c Intracellular trafficking of DG9-PMO in del45 myoblasts, myotubes, and healthy cardiomyocytes in presence of different endocytosis inhibitors; NaN3 and 2′-Deoxy-D-Glucose for ATP-depletion, chlorpromazine (CPZ) for clathrin-mediated endocytosis inhibition, nystatin and genistein for caveolae-mediated endocytosis inhibition, and 5-(N-ethyl-N-isopropyl) amiloride (EIPA) for macropinocytosis inhibition (n = 3). Statistical analysis: One-way ANOVA with Dunnet’s test against non-inhibited. Not significant, P > 0.05 (not shown). Error: SEM. Source data are provided as a Source Data file.

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  Fig. 2. Skeletal muscle improvement after DG9-PMO treatment. a Eight week-old male hDMDdel45;mdx mice were treated retro-orbitally four times once/week with either saline, DG9-PMO, or PMO at 30 mg/kg bw. Saline-treated WT served as control. Functional tests were conducted before and 1 week after treatment. Blood and urine samples were collected 10 days post-treatment. Tissues were collected 2 weeks after the last injection. All mice received a 15 mg/kg subcutaneous injection of β-isoproterenol and 1% Evans blue dye intraperitoneally 24 h before sacrifice. b Forelimb and total grip strength of individual groups of mice normalized to their body weight after treatment. Changes in endurance and coordination compared to baseline measured using treadmill and rotarod (n = 4–6). c Quantification of exon 44 skipping by RT-PCR (left) and dystrophin restoration by western blot (right) in various skeletal muscles after treatment (n = 5). Dystrophin (Leica, NCL-DYS1) restoration presented relative to wild-type control after normalization with desmin (Abcam, ab8592). Statistical analysis: One-way ANOVA with Tukey’s test. Not significant, P > 0.05 (not shown). Error: SEM (b, c). d Representative…

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  Fig. 3. DG9-PMO provides cardio-protection after β-isoproterenol induced cardiac damage by restoring dystrophin production. a RT-PCR of exon 44 skipping and western blot (NCL-Dys1) results post-treatment in the heart. Proteins were loaded at 40 µg for NT and treated along with indicated percentages for the WT. Desmin (ab8592) and MyHC serve as loading controls. Quantification is shown on the right relative to wild-type control after normalization with desmin (n = 4–5). b Representative immunofluorescence images for dystrophin (green, ab15277) in the heart. Percentage of dystrophin-positive fibers calculated in relation to laminin. Evans blue dye uptake as a result of β-isoproterenol induced cardiac damage shown in red. Quantification is shown on the right (n = 3–5). c 10 week-old male hDMDdel45;mdx mice were treated with 1.5 mg/kg of β-isoproterenol (subcutaneous) every day for 14 days. These mice also received two 30 mg/kg doses of DG9-PMO or saline once/week retro-orbitally. Isoproterenol-treated WT served as control. Echocardiogram tests were conducted before and 1 day after treatment. Blood samples were collected after the treatment. d Changes in fractional shortening and ejection…

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  Fig. 4. Normalized liver and kidney biomarker levels with reduced inflammation in skeletal muscles post-treatment. a Analysis of different liver-specific (aspartate aminotransferase, alanine transaminase, creatine kinase) and kidney-specific (blood urea nitrogen) biomarkers from serum samples collected 10 days post-treatment. Kidney injury molecule 1 (KIM-1) was analyzed from urine samples collected at the same time point (n = 4–5). b Representative immunofluorescence images for CD68+ cells (green, MCA1957T) with DAPI staining. Quantification shown on the right (n = 3–5). Statistical analysis: One-way ANOVA with Tukey’s test. Not significant, P > 0.05 (not shown). Error: SEM. Scale 100 µm. Source data are provided as a Source Data file.

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  Fig. 5. RNA sequencing analysis of DG9-PMO therapeutic efficacy. a Venn diagram showing the number of unique and overlapping differentially expressed genes (DEGs) in different treatment groups for the TA and heart; NT compared to WT, DG9-PMO treated compared to NT and WT (n = 4). b Regression analysis of the log2(fold-change) values of overlapping DEGs in hDMDdel45;mdx mice and DMD patient TA muscles (Nieves-Rodriguez et al.36) or iPSC-derived cardiomyocytes (Kamdar et al.37). c Heatmaps displaying the expression changes in the TA and heart as a result of the treatment. Top 50 DEGs, common for both humans and mice, with the highest statistical significance in dystrophic mice compared to WT are presented. High expression indicated by red with blue indicating low expression. Dots represent significantly altered gene expression (P < 0.05). Statistical analysis: Two-sided Wald test with Benjamini-Hochberg multiple testing correction. d Selected top GO terms enriched in up and downregulated genes after DG9-PMO treatment in reference to NT. Analysis was done using Metascape38.

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  Fig. 6. Comparison between DG9- and R6G-PMO. a Schematic diagram of the treatment regimen. Eight week-old male hDMDdel45;mdx mice were treated retro-orbitally twice, once/week, with DG9-PMO or R6G-PMO at 30 mg/kg (equivalent to 2190 nmol/kg for DG9-PMO, 2689 nmol/kg for R6G-PMO). Tissues were collected 1 week post-treatment. b Western blot (NCL-Dys1) images post-treatment in TA and heart. Proteins were loaded at 40 µg for NT and treated along with indicated percentages for the WT. Desmin (ab8592) serves as loading control. Quantification is shown on the right relative to wild-type control after normalization with desmin (n = 5). c RT-PCR of exon 44 skipping in TA and the heart. Quantification is shown at the bottom (n = 5). d Representative immunofluorescence images for dystrophin (ab15277) in TA and heart muscles. Percentage of dystrophin-positive fibers calculated in relation to laminin. Scale 200 µm. Statistical analysis: unpaired two-tailed t-test. Error: SEM. Source data are provided as a Source Data file.