Abstract
Antisense oligonucleotide (AO)–mediated exon-skipping therapy is one of the most promising therapeutic strategies for Duchenne Muscular Dystrophy (DMD) and several AO chemistries have been rigorously investigated. In this report, we focused on the effect of 2′-O-methoxyethyl oligonucleotides (MOE) on exon skipping in cultured mdx myoblasts and mice. Efficient dose-dependent skipping of targeted exon 23 was achieved in myoblasts with MOE AOs of different lengths and backbone chemistries. Furthermore, we established that 25-mer MOE phosphorothioate (PS) AOs provided the greatest exon-skipping efficacy. When compared with 2′O methyl phosphorothioate (2′OmePS) AOs, 25-mer MOE (PS) AOs also showed higher exon-skipping activity in vitro and in mdx mice after intramuscular injections. Characterization of uptake in vitro corroborated with exon-skipping results, suggesting that increased uptake of 25-mer MOE PS AOs might partly contribute to the difference in exon-skipping activity observed in vitro and in mdx mice. Our findings demonstrate the substantial potential for MOE PS AOs as an alternative option for the treatment of DMD.
Highlights
Duchenne muscular dystrophy (DMD) is a lethal muscle degenerative disease that arises from mutations, typically large deletions, in the DMD gene resulting in out-of-frame dystrophin transcripts and in the lack of functional dystrophin protein
We demonstrated that methoxyethyl oligonucleotides (MOE) (PS) Antisense oligonucleotide (AO) can effectively induce exon-skipping better than 29OMePS AOs both in vitro and in mdx mice and that the increased exon-skipping efficiency is probably due to increased cellular uptake
Whereas for MOE25(PS), about 95% exon-skipping was achieved at 500 nM and no significant difference was detected between 500 nM and 1 mM, suggesting that MOE25(PS) AOs reached saturation at the concentration of 500 nM (Fig.1A–B)
Summary
Duchenne muscular dystrophy (DMD) is a lethal muscle degenerative disease that arises from mutations, typically large deletions, in the DMD gene resulting in out-of-frame dystrophin transcripts and in the lack of functional dystrophin protein. Antisense oligonucleotides (AOs) are short single-stranded nucleic acids capable of effecting splice correction of aberrant disease-related pre-mRNA transcripts in order to restore their function [1]. Such AOs have been shown to correct aberrant outof-frame dystrophin transcripts via the exclusion of specific dystrophin exons, thereby restoring the open reading frame to generate a shortened but functional dystrophin protein product [2]. By conjugating CPPs to PMO, the exonskipping efficacy and level of dystrophin expression can be significantly enhanced [19,20], the reported toxicity profiles of CPPs may limit their clinical use
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