Combined Treatment with Peptide-Conjugated Phosphorodiamidate Morpholino Oligomer-PPMO and AAV-U7 Rescues the Severe DMD Phenotype in Mice

Abstract

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by an absence of the dystrophin protein, which is essential for muscle fiber integrity. Among the developed therapeutic strategies for DMD, the exon-skipping approach corrects the frameshift and partially restores dystrophin expression. It could be achieved through the use of antisense sequences, such as peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) or the small nuclear RNA-U7 carried by an adeno-associated virus (AAV) vector. AAV-based gene therapy approaches have potential for use in DMD treatment but are subject to a major limitation: loss of the AAV genome, necessitating readministration of the vector, which is not currently possible, due to the immunogenicity of the capsid. The PPMO approach requires repeated administrations and results in only weak cardiac dystrophin expression. Here, we evaluated a combination of PPMO- and AAV-based therapy in a mouse model of severe DMD. Striking benefits of this combined therapy were observed in striated muscles, with marked improvements in heart and diaphragm structure and function, with unrivalled extent of survival, opening novel therapeutic perspectives for patients.

Graphical abstract

Figure 1

Combined PPMO + AAV-U7 Treatment Induces Efficient Exon Skipping and Dystrophin Protein Restoration in the Heart and Diaphragm (A) Schematic diagram of the treatments administered to the dKO mice: PPMO, AAV-U7, and PPMO + AAV-U7. IV, intravenous; IP, intraperitoneal; D, day; W, week. (B) Quantification of exon 23 skipping in heart, diaphragm, and tibialis anterior (TA) from 22-week-old dKO mice treated with AAV-U7 or with PPMO + AAV-U7. Each dot represents one mouse. The data shown are means ± SEM for 10 mice per group. The significance of differences was determined by one-way ANOVA. (C) Representative immunoblots showing dystrophin levels in the heart, diaphragm, and TA from 22-week-old treated and wild-type (WT) mice; α-actinin is shown as a loading control. The results shown are mean ± SEM for 10 mice per group. The significance of differences was determined by one-way ANOVA. (D) Micrographs showing dystrophin and β-sarcoglycan labeling in the heart, diaphragm (Dia), and TA of 22-week-old WT and treated dKO mice. Scale bars, 100 μm.

Figure 2

The Combined Approach Extends Survival in dKO Mice (A) Survival for untreated and treated dKO mice. The survival curves are significantly different, p < 0.0001, Mantel-Cox log-rank test. (B) Gross morphology of 22-week-old dKO mice treated with PPMO, AAV-U7, or PPMO + AAV-U7. Comparison of body weight between mice from the different groups during the course of the disease. The data shown are as means ± SEM (n = 10 per group), and significance was determined by one-way ANOVA. Significant differences relative to the WT are indicated. (C and D) Ratios of heart or diaphragm (C) or skeletal (D) muscle weight to body weight for 22-week-old WT and treated dKO mice. The data shown are mean ± SEM for 10 mice per group. Significance was determined by one-way ANOVA.

Figure 3

The Combined Approach Protects dKO Muscles from Fibrosis and Inflammation (A) Representative micrographs of hematoxylin-eosin- and sirius red-stained sections of heart, diaphragm, and TA from the indicated groups of mice. Scale bars, 100 μm. (B) Fibrosis calculated as a percentage of the total area with ImageJ software, from sirius red-stained sections from 22-week-old mice. The data shown are means ± SEM of at least six mice per group, and significance was determined by one-way ANOVA. (C and D) Fiber size distribution (C) and percentage of centrally nucleated fibers (D) calculated with MuscleJ software after laminin fluorescence staining for the diaphragm (top panel) and TA (bottom panel). The data shown are mean ± SEM for at least 10 mice per group, and significance was determined by one-way ANOVA (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 relative to WT mice; $p < 0.05, $$p < 0.01, $$$p < 0.001 relative to other treated mice).

Figure 4

The Combined Approach Restores Normal Cardiac and Respiratory Functions and Improves Skeletal Muscle Function (A) RT-quantitative real-time PCR analysis of Myh7 and Nppa expression in the hearts of 9-week-old WT, mdx, and untreated dKO mice and 22-week-old WT mice and dKO mice treated with PPMO, AAV-U7, or PPMO + AAV-U7. The data shown are mean ± SEM for at least 10 mice per group, and significance was determined by one-way ANOVA. (B) Micrographs showing connexin 43 and desmoplakin labeling in the heart for 9-week-old WT and dKO mice. 4′,6-Diamidino-2-phenylindole (DAPI) counterstaining (blue) of the nuclei. Arrows indicate the lateralization of connexin 43. Scale bars, 10 μm. The colocalization of connexin 43 and desmoplakin was quantified for the various groups of mice at the age indicated. Significance was determined by one-way ANOVA. (C) Respiratory parameters for 9-week-old WT (n = 12), mdx (n = 13), and dKO (n = 13) mice; 22-week-old WT (n = 12), PPMO-treated (n = 10), AAV9-U7-treated (n = 10), and PPMO + AAV9-U7-treated (n = 10) dKO mice; and 40-week-old WT (n = 6) and PPMO + AAV9-U7-treated (n = 4) dKO mice fR, respiratory frequency; V_T, tidal volume; V_E, minute ventilation. The data shown are means ± SEM. Significance was determined by one-way ANOVA for 22-week-old mice and with unpaired t tests for 40-week-old mice. (D) Specific maximal force and force drop resulting from the injury induced by 10 lengthening contractions of the TA for 22-week-old WT, mdx, and treated dKO mice. The data shown are mean ± SEM for at least 10 mice per group, and significance was determined by one-way ANOVA.

Figure 5

Long-Term Combined PPMO + AAV-U7 Treatment (A) Quantification of the number of vector genomes in the heart, diaphragm, and tibialis anterior (TA) of AAV-U7- and PPMO + AAV-U7-treated mice. The data shown are mean ± SEM for 10 mice per group at 22 weeks of age and four mice at 40 weeks of age. Significance was determined by one-way ANOVA. (B) Representative immunoblots and quantification of dystrophin and α-actinin in striated muscles from WT (dark blue) and PPMO + AAV-U7-treated dKO mice (light blue) at 40 weeks of age. The data shown are means ± SEM for 6 WT and 4 PPMO + AAV-treated dKO mice. Significance was determined in unpaired t tests. (C) Representative cropped immunoblots of dystrophin and α-actinin for the heart, diaphragm, and TA of PPMO + AAV-U7-treated dKO mice at 22 and 40 weeks of age. (D) Representative immunoblots of dystrophin and α-actinin for the heart, diaphragm, and TA of WT (50 weeks old) mice and dKO mice treated with PPMO (about 45 weeks old) or PPMO + AAV-U7 (about 52 weeks old) at the humane endpoint. (E) Staining with hematoxylin and eosin and sirius red of heart, diaphragm (Dia), and TA sections from WT mice and from PPMO- and PPMO + AAV-U7-treated dKO mice at about 50, 45, and 52 weeks of age, respectively.

Figure 6

Schematic Diagram of the Benefits of the Different Treatments in Striated Muscles