In-frame Dystrophin Following Exon 51-Skipping Improves Muscle Pathology and Function in the Exon 52–Deficient mdx Mouse

Abstract

A promising therapeutic approach for Duchenne muscular dystrophy (DMD) is exon skipping using antisense oligonucleotides (AOs). In-frame deletions of the hinge 3 region of the dystrophin protein, which is encoded by exons 50 and 51, are predicted to cause a variety of phenotypes. Here, we performed functional analyses of muscle in the exon 52-deleted mdx (mdx52) mouse, to predict the function of in-frame dystrophin following exon 51-skipping, which leads to a protein lacking most of hinge 3. A series of AOs based on phosphorodiamidate morpholino oligomers was screened by intramuscular injection into mdx52 mice. The highest splicing efficiency was generated by a two-oligonucleotide cocktail targeting both the 5' and 3' splice sites of exon 51. After a dose-escalation study, we systemically delivered this cocktail into mdx52 mice seven times at weekly intervals. This induced 20-30% of wild-type (WT) dystrophin expression levels in all muscles, and was accompanied by amelioration of the dystrophic pathology and improvement of skeletal muscle function. Because the structure of the restored in-frame dystrophin resembles human dystrophin following exon 51-skipping, our results are encouraging for the ongoing clinical trials for DMD. Moreover, the therapeutic dose required can provide a suggestion of the theoretical equivalent dose for humans.

Parental attitudes toward newborn screening for Duchenne/Becker muscular dystrophy and spinal muscular atrophy

“…These AOs are designed to bind to a specific region of mRNA post-transcription to alter splicing of the mRNA such that the mature transcript has an in-frame deletion, thus rescuing a dystrophin protein that retains both N-and Cterminals (Fragall, 2011;Manzur, 2009). Depending on the dystrophin gene deletion location and its effect on reading frame, skipping an adjacent exon can restore the reading frame and potentially modify a DMD phenotype to enable a clinical course of a milder BMD phenotype (Nakamura, 2009;Aoki, 2010). Based on an analysis of the prevalence of specific deletions, skipping exon 51 would rescue the highest percentage of DMD deletions, and clinical trials testing exon 51 skipping strategies are currently showing promise (Lu, 2011;Brolin, 2011;.…”

Bodywide skipping of exons 45–55 in dystrophic mdx52 mice by systemic antisense delivery

“…Total RNA from the muscles of WT, untreated, or mdx52 mice were extracted as previously described (7). Two hundred nanograms of RNA template was used for a 20-μL RT-PCR using a QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer's instructions.…”

“…Western blot analysis was performed as previously described (7). Two to 20 μg of protein from the TA muscle of a WT mouse as a positive control, 20 μg of protein from the TA muscle of untreated mdx52 as a negative control, and 20 μg of protein from the muscles of treated mdx52 mice were loaded onto a 5-15% (wt/vol) XV Pantera gel (DRC).…”

Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD)

“…Recently, other animal models have been introduced such as dystrophin/utrophin double knockout mice, 15 humanized DMD mice, 16 mdx52 mice (carrying a deletion of exon 52 in murine DMD), 17 4CV mice (carrying a nonsense mutation in exon53), 18 and canine DMD model (mutation in exon 7). 19 These transgenic mice provide indispensable tools for investigating the exon skipping strategies targeting above…”