Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles

Abstract

Antisense oligonucleotide-mediated alternative splicing has great potential for treatment of Duchenne muscular dystrophy (DMD) caused by mutations within nonessential regions of the dystrophin gene. We have recently shown in the dystrophic mdx mouse that exon 23, bearing a nonsense mutation, can be skipped after intramuscular injection of a specific 2'-O-methyl phosphorothioate antisense oligoribonucleotide (2OMeAO). This skipping created a shortened, but in-frame, transcript that is translated to produce near-normal levels of dystrophin expression. This expression, in turn, led to improved muscle function. However, because DMD affects muscles body-wide, effective treatment requires dystrophin induction ideally in all muscles. Here, we show that systemic delivery of specific 2OMeAOs, together with the triblock copolymer F127, induced dystrophin expression in all skeletal muscles but not in cardiac muscle of the mdx dystrophic mice. The highest dystrophin expression was detected in diaphragm, gastrocnemius, and intercostal muscles. Large numbers of fibers with near-normal level of dystrophin were observed in focal areas. Three injections of 2OMeAOs at weekly intervals enhanced the levels of dystrophin. Dystrophin mRNA lacking the targeted exon 23 remained detectable 2 weeks after injection. No evidence of tissue damage was detected after 2OMeAO and F127 treatment either by serum analysis or histological examination of liver, kidney, lung, and muscles. The simplicity and safety of the antisense protocol provide a realistic prospect for treatment of the majority of DMD mutations. We conclude that a significant therapeutic effect may be achieved by further optimization in dose and regime of administration of antisense oligonucleotide.

Figures

Fig. 1.

Restoration of dystrophin expression in skeletal and cardiac muscles. (Left) Muscles of control mdx mouse. (Center) Muscles of mdx mouse after single tail-vein injection of the 2OMeAOs. (Right) Muscles of mdx mouse after triple tail-vein injections of the 2OMeAO at weekly intervals. TA, tibialis anterior. Muscles were examined by immunohistochemistry with polyclonal antibody P7 to dystrophin and detected with Alexa Fluor 594-labeled goat anti-rabbit Igs. Red staining on fiber membrane shows dystrophin expression. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue).

Fig. 2.

The number of positive fibers expressing membrane-localized dystrophin 2 weeks after the 2OMeAO treatments. mdx mice were treated at the age of 3 weeks (A), 6 weeks (B), and 6 months (C). Control, mdx injected with saline; TA, tibialis anterior; AO, 2OMeAO; Triple injections, injections at weekly intervals. The mean percentages of dystrophin-positive fibers in the 6-week-old group were 6%, 7.2%, 8.6%, and 8.2% in the AO-treated TA, quadriceps, gastrocnemius and biceps, respectively, in comparison with the 1.8%, 1.4%, 2.0%, and 1.7% in the control mice. The percentages were 12.5%, 9.4%, 16.2%, and 13.9% after triple injections. The percentages in the 6-month-old group were 8.9%, 11.4%, 7.8%, and 9.5% in the AO-treated TA, quadriceps, gastrocnemius, and biceps, respectively, in comparison with 2.8%, 3.1%, 2.6%, and 3.2% in the control mice. The percentage of dystrophin-positive fibers in the 3-week-old group was <1% in all four muscles except in the AO-treated quadriceps, which is 1.2%. The larger percentage of positive fibers when compared with the percentage of protein levels (Fig. 3A) may be attributed to the presence of revertant fibers, the lower-than-normal levels of the protein in most dystrophin-positive fibers, and the use of maximum numbers of dystrophin-positive fibers for each muscle sample. Sections were stained with polyclonal antibody P7 to dystrophin and detected with Alexa Fluor 594-labeled goat anti-rabbit Igs. (*, P < 0.05, ANOVA test; n = 4–6 mice).

Fig. 3.

Detections of dystrophin protein by Western blot and truncated dystrophin mRNA by RT-PCR 2 weeks after the triple tail-vein injections of the 2OMeAO. (A) No visible difference in the size of the dystrophins between muscles treated with the 2OMeAO and muscle from the normal C57BL/10 mouse. Up to 5% of normal wild-type concentration of dystrophin was detected in muscles of gastrocnemius, intercostal, diaphragm, and abdominal muscles. (B) mRNA with exon 23 exclusion was clearly demonstrated in gastrocnemius, intercostal, diaphragm, quadriceps, and abdominal muscles. Positive control, mRNA from C2C12 cell culture treated with the same 2OMeAOs and known to contain transcripts with single exon 23 exclusion and both exon 22 and 23 exclusion. Negative control, without template. The major bands indicated by the boxed numbers of 22, 23, and 24 (B Left) are the RT-PCR products (901 base pairs) representing full-length dystrophin; the bands below indicated by the boxed numbers of 22 and 24 are the products (688 base pairs) representing mRNA with exon 23 skipped. One smaller band in the positive control lane is the product from mRNA with both exon 22 and 23 skipped.

Fig. 4.

Restoration of dystrophin-associated proteins. (Left) Muscle of control mdx mouse. (Right) Muscle of mdx mouse after triple tail-vein injections of the 2OMeAO at weekly intervals. Membrane localized α-sarcoglycan, β-dystroglycan and neuronal nitric oxide synthase (nNOS) are detected in the fibers expressing dystrophin after the 2OMeAO treatment. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue).

Fig. 5.

Measurements of serum enzymes and electrolytes. Na, sodium; K, potassium; Creat, creatinine; ALT, alanine transaminase; AST, aspartime transaminase. Significant differences (marked by P values) are observed only between mdx and C57BL/10 mice (potassium, 16.4 ± 0.9 vs. 8.66 ± 0.41, P = 0.0079; creatinine, 39.82 ± 1.79 vs. 28.4 ± 1.21, P = 0.0005; urea, 8.53 ± 0.98 vs. 7.94 ± 0.22, P = 0.028). No significant difference was observed between treated and untreated mdx or C57BL/10 mice (ANOVA test; n = 4–6 mice).