Exon skipping restores dystrophin expression, but fails to prevent disease progression in later stage dystrophic dko mice

Abstract

Antisense therapy with both chemistries of phosphorodiamidate morpholino oligomers (PMOs) and 2'-O-methyl phosphorothioate has demonstrated the capability to induce dystrophin expression in Duchenne muscular dystrophy (DMD) patients in phase II-III clinical trials with benefit in muscle functions. However, potential of the therapy for DMD at different stages of the disease progression is not understood. In this study, we examined the effect of peptide-conjugated PMO (PPMO)-mediated exon skipping on disease progression of utrophin-dystrophin-deficient mice (dko) of four age groups (21-29, 30-39, 40-49 and 50+ days), representing diseases from early stage to advanced stage with severe kyphosis. Biweekly intravenous (i.v.) administration of the PPMO restored the dystrophin expression in nearly 100% skeletal muscle fibers in all age groups. This was associated with the restoration of dystrophin-associated proteins including functional glycosylated dystroglycan and neuronal nitric synthase. However, therapeutic outcomes clearly depended on severity of the disease at the time the treatment started. The PPMO treatment alleviated the disease pathology and significantly prolonged the life span of the mice receiving treatment at younger age with mild phenotype. However, restoration of high levels of dystrophin expression failed to prevent disease progression to the mice receiving treatment when disease was already at advanced stage. The results could be critical for design of clinical trials with antisense therapy to DMD.

Figure 1

Effect of PPMOE23 treatment on disease phenotype and life span of the dko mice. Untreated, control dko group. Numbers represent the age group of dko mice when the treatment starts. (a) Life spans of dko mice from untreated and treated groups. (b) Mice body weight changes during the treatment from untreated and treated groups. (c) Mice body size comparison, appearance and abnormal limb contraction of 70-day-old mice from untreated, 20–29D and 40–49D treated groups. (d) Onset of abnormal walking limb posture from untreated and treated groups. (e) Percentages of mice with eye infections from untreated and treated groups. (f) Kyphosis detected by X-ray images from untreated and treated groups. (g) Kyphosis index analysis from untreated and treated groups. (h) Onset of kyphosis from untreated and treated groups. Mean ± s.e.m., n = 10. Statistical differences between treatment groups and control groups were evaluated by Student’s t-test. *P = 0.05.

Figure 2

Restoration of dystrophin expression in all groups of dko mice treated with 15 mg kg⁻¹ PPMOE23 biweekly. (a) Dystrophin expression in skeletal muscles and the heart in all treated groups. (b) Western blotting demonstrates the levels of dystrophin expression in the TA (tibialis anterior) muscle, diaphragm and the heart. Three samples for each treated group. Dystrophin detection is shown in the upper three lanes, and α-actin is used as a loading control from TA muscles. The low panel shows the quantification of dystrophin levels from TA, diaphragm and the heart of the treated groups with corresponding muscles of C57 mice as 100% (labeled as 1). (c) Detection of dystrophin exon 23 skipping by RT-PCR. E22-E23-E24 and E22-E24 representing normal mRNA and the mRNA with exon 23 skipped, respectively.

Figure 3

Immunohistochemistry for membrane nNOS (with rabbit-anti-nNOS, Millipore), functionally glycosylated α-dystroglycan (with IIH6, Millipore) and β1-intergin (with goat-anti-β1 R…D system) expression after PPMOE23 treatment in tibialis anterior muscles of dko mice. Untreated, control group dko mice. Numbers represent the age group of dko mice when the treatment starts.

Figure 4

Effect of PPMOE23 treatment on muscle pathology and serum components. (a) Hematoxylin and eosin staining for muscles, the kidney and the liver. No pathologic change of the liver and the kidney was observed in the PPMO-treated groups. Numbers represent the age group of dko mice when the treatment starts. TA, tibialis anterior muscle. (b) Collagen deposition in diaphragms detected by Masson’s trichrome staining. The degree of collagen deposition is directly related to the delayed treatment. (c) Serum tests. Creatine kinase levels are most significantly reduced in the PPMOE23-treated 20–29D group dko mice, but not different in 50+D group when compared with the untreated group. Similar improvement in reduction of total bilirubin, ALT, ALP is also observed in the three younger treated groups. Mean±s.e.m., n = 10. Statistical differences between treatment groups and control groups were evaluated by Student’s t-test. ALP, alkaline phosphatase; ALT, alanine transaminase.