Prednisolone treatment does not interfere with 2'-O-methyl phosphorothioate antisense-mediated exon skipping in Duchenne muscular dystrophy

Abstract

In Duchenne muscular dystrophy (DMD), dystrophin deficiency leading to progressive muscular degeneration is caused by frame-shifting mutations in the DMD gene. Antisense oligonucleotides (AONs) aim to restore the reading frame by skipping of a specific exon(s), thereby allowing the production of a shorter, but semifunctional protein, as is found in the mostly more mildly affected patients with Becker muscular dystrophy. AONs are currently being investigated in phase 3 placebo-controlled clinical trials. Most of the participating patients are treated symptomatically with corticosteroids (mainly predniso[lo]ne) to stabilize the muscle fibers, which might affect the uptake and/or efficiency of AONs. Therefore the effect of prednisolone on 2'-O-methyl phosphorothioate AON efficacy in patient-derived cultured muscle cells and the mdx mouse model (after local and systemic AON treatment) was assessed in this study. Both in vitro and in vivo skip efficiency and biomarker expression were comparable between saline- and prednisolone-cotreated cells and mice. After systemic exon 23-specific AON (23AON) treatment for 8 weeks, dystrophin was detectable in all treated mice. Western blot analyses indicated slightly higher dystrophin levels in prednisolone-treated mice, which might be explained by better muscle condition and consequently more target dystrophin pre-mRNA. In addition, fibrotic and regeneration biomarkers were normalized to some extent in prednisolone- and/or 23AON-treated mice. Overall these results show that the use of prednisone forms no barrier to participation in clinical trials with AONs.

FIG. 1.

Effect of prednisolone on antisense oligonucleotide-mediated exon skipping in vitro and intramuscularly in the mdx mouse. Means are shown for each group. Error bars represent the standard deviation. (A) In two patient cell lines no effect on skipping percentages on addition of prednisolone was seen (n=12 per condition). For 53914.1 cells (deletion of exon 52) AONs against exon 51 were used, and for 589.2 cells (deletion of exons 51–55) AONs against exon 50 were used. (B) Prednisolone did not affect exon skipping after intramuscular injection of 23AON into the gastrocnemius of mdx mice. Per group, eight muscles were analyzed.

FIG. 2.

Systemic treatment with prednisolone and/or 23AON. Shown is the effect on body weight of all treatments and antisense oligonucleotide-mediated exon skipping and 23AON biodistribution after cotreatment with prednisolone or saline. Per group, eight mice were analyzed. Means are shown for each group. Error bars represent the standard deviation. (A) Body weight was decreased in both prednisolone-treated groups compared with both saline- and 23AON alone-treated mice (p<0.01). (B) RT-PCR analysis for the tibialis anterior. Wild-type product consists of 334 base pairs and exon 23 skipping results in a 122-base pair product. AON, antisense oligonucleotide; ctrls, controls; S, saline; P, prednisolone. (C) No difference in exon skip percentages between saline and prednisolone cotreatment with 23AONs was seen. Exon skip percentages were determined by DNA 1000 LabChip analysis on an Agilent 2100 bioanalyzer. (D) Biodistribution analysis showed a small decrease in 23AON uptake in the quadriceps and diaphragm, but not in the heart. **p<0.01 compared with 23AON alone-treated mice.

FIG. 3.

Dystrophin expression detected by immunofluorescence staining and Western blot analysis. Per group, eight mice were analyzed. Means are shown for each group. Error bars represent the standard deviation. (A) Representative example of Western blot analysis (gastrocnemius muscle), showing low levels of dystrophin protein (top) in all 23AON-treated mice. No band was detected in control mice. Myosin (bottom) was used as loading control. (B) Quantification of dystrophin protein expression by Western blot showed a slight increase in protein levels in 23AON-treated mice, which was significant for the gastrocnemius muscle, but not for the diaphragm. *p<0.05 compared with 23AON alone-treated mice. (C) Immunofluorescence staining with dystrophin antibody in the gastrocnemius muscle showed dystrophin expression above background levels in both 23AON-treated groups, but no difference between prednisolone- and saline-treated mice. **p<0.01 compared with saline-treated mice

FIG. 4.

Expression of biomarker levels measured by quantitative PCR. Per group, eight mice were analyzed. Means are shown for each group, corrected for GAPDH or 5S expression. Values are expressed relatively to tibialis anterior levels of saline-treated mice. Error bars represent the standard deviation. Immunological markers CD68 (A) and Lgals3 (B), fibrotic markers biglycan (C) and Lox (D), early regeneration markers MyoD (E) and myogenin (F), late regeneration marker MRF4 (G) and miR-31 (H) were measured. miR-31 was determined only in the diaphragm (values expressed relatively to saline-treated mice) *p<0.05 **p<0.01 compared with saline-treated mice.