Exon skipping-mediated dystrophin reading frame restoration for small mutations

Abstract

Exon skipping using antisense oligonucleotides (AONs) has successfully been used to reframe the mRNA in various Duchenne muscular dystrophy patients carrying deletions in the DMD gene. In this study we tested the feasibility of the exon skipping approach for patients with small mutations in in-frame exons. We first identified 54 disease-causing point mutations. We selected five patients with nonsense or frameshifting mutations in exons 10, 16, 26, 33, and 34. Wild-type and mutation specific 2'OMePS AONs were tested in cell-free splicing assays and in cultured cells derived from the selected patients. The obtained results confirm cell-free splicing assay as an alternative system to test exon skipping propensity when patients' cells are unavailable. In myogenic cells, similar levels of exon skipping were observed for wild-type and mutation specific AONs for exons 16, 26, and 33, whereas for exon 10 and exon 34 the efficacy of the AONs was significantly different. Interestingly, in some cases skipping efficiencies for mutated exons were quite dissimilar when compared with previous reports on the respective wild-type exons. This behavior may be related to the effect of the mutations on exon skipping propensity, and highlights the complexity of identifying optimal AONs for skipping exons with small mutations.

Antisense-Induced Messenger Depletion Corrects a COL6A2 Dominant Mutation in Ullrich Myopathy

“…A 2¢OMePS-modified RNA AON [referred to as P221(T)AON] was designed, according to published guidelines (Aartsma-Rus, 2012), to target this T allele (Supplementary Fig. S1A; supplementary data are available online at www.liebertonline.com/hum); synthesis and purification were as previously described (Spitali et al, 2009). Various AON doses and timings were tested (see Supplementary Table S1).…”

“…We adopted exon skipping, exploring the efficacy of an SNP-specific 2¢OMePS-AON to induce out-of-frame skipping and consequent allele degradation, as a corrective approach for a COL6A2 gene dominant negative mutation. This strategy was based on the following evidence: (1) 2¢OMePS-AON sequence specificity and efficacy in inducing exon removal have been largely demonstrated in humans (Spitali et al, 2009); (2) COL6 gene transcripts with nonsense mutations almost invariably undergo RNA nonsense-mediated degradation, and quantification of transcripts has been proposed as a tool for pinpointing the mutated gene (Allamand et al, 2011); (3) COL6 genes are highly polymorphic and several common SNPs are known, which, if cistronic with pathogenic mutations, can be specifically targeted by AONs to induce transcript depletion in large patient populations;…”

The Role of Stem Cells in Muscular Dystrophies

“…Golden Retriever Muscular Dystrophy (GRMD) dogs carry a splice site mutation in intron 6 that excludes exon 7 from the mRNA transcript and they are an excellent model for testing the efficacy and efficiency of double-exon skipping [113]. Multiple exon skipping could restore open reading frame in >80% both of deletion and nonsense mutations in the dystrophin gene [114]. However it has been proposed that a cocktail of antisense oligonucleotides covering exons 45-55, could be used in a high percentage of asymptomatic or mild BMD clinical phenotypes and it could be potentially applicable to 63% of dystrophic patients [115].…”

Antisense oligonucleotide corrects splice abnormality in hereditary myopathy with lactic acidosis

“…AOs have been used in myoblasts to modify the effects of deleterious frameshifting mutations in Duchenne muscular dystrophy (DMD) (Aartsma-Rus et al, 2003), or against PTCs in both the mdx mouse (Mann et al, 2002) and patients with DMD (Spitali et al, 2009). Reading frame can be restored or PTCs eliminated by using AOs to induce skipping of flanking exons; the result is a protein that contains a deletion, but which is partially functional.…”