Abstract
Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON.
Highlights
Duchenne muscular dystrophy (DMD) is a severe X-linked muscle-wasting disorder affecting 1 in 5,000 newborn boys.[1,2] DMD is caused by out-of-frame or nonsense mutations in the DMD gene that lead to a truncated, nonfunctional dystrophin protein
Dystrophin is an important shock-absorbing protein in muscle and without it, muscles are damaged. Restoration of the reading frame in DMD patients would in theory allow the production of a shorter, but partly functional dystrophin protein as seen in less severely affected Becker muscular dystrophy patients.[3,4]
In vitro evaluation To test whether 2’-F phosphorothioate (2FPS) antisense oligonucleotides (AONs) are capable of inducing dystrophin exon skipping, human control myotube cultures were transfected with 100–500 nmol/l of several 2FPS AONs and their isosequential 2’-O-methyl phosphorothioate (2OMePS) counterparts.[14]
Summary
Duchenne muscular dystrophy (DMD) is a severe X-linked muscle-wasting disorder affecting 1 in 5,000 newborn boys.[1,2] DMD is caused by out-of-frame or nonsense mutations in the DMD gene that lead to a truncated, nonfunctional dystrophin protein. Dystrophin is an important shock-absorbing protein in muscle and without it, muscles are damaged. Restoration of the reading frame in DMD patients would in theory allow the production of a shorter, but partly functional dystrophin protein as seen in less severely affected Becker muscular dystrophy patients.[3,4] This can be achieved with antisense oligonucleotides (AONs) that target and induce skipping of specific exons during pre-mRNA splicing.[5,6] Exon skipping AONs are thought to act by sterically hindering splicing factors in the recognition of the exon and/or splicing sites. Two different AON chemistries, phosphorodiamidate morpholino oligonucleotides and 2’-O-methyl phosphorothioate (2OMePS), are currently in clinical development for exon skipping in DMD.[8,9,10,11,12]
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