Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells

Abstract

As small molecule drugs for Duchenne muscular dystrophy (DMD), antisense oligonucleotides (AONs) have been shown to restore the disrupted reading frame of DMD transcripts by inducing specific exon skipping. This allows the synthesis of largely functional Becker muscular dystrophy (BMD)-like dystrophins and potential conversion of severe DMD into milder BMD phenotypes. Thus far we have used 2 0 -O-methyl phosphorothioate (2OMePS) AONs. Here, we assessed the skipping efficiencies of different AON analogs containing morpholino-phosphorodiamidate, locked nucleic acid (LNA) or peptide nucleic acid (PNA) backbones. In contrast to PNAs and morpholinos, LNAs have not yet been tested as splice modulators. Compared to the most effective 2OMePS AON directed at exon 46, the LNA induced higher skipping levels in myotubes from a human control (85 versus 20%) and an exon 45 deletion DMD patient (98 versus 75%). The morpholinoinduced skipping levels were only 5-6%, whereas the PNA appeared to be ineffective. Further comparative analysis of LNA and 2OMePS AONs containing up to three mismatches revealed that LNAs, while inducing higher skipping efficiencies, show much less sequence specificity. This limitation increases the risk of adverse effects elsewhere in the human genome. Awaiting further improvements in oligochemistry, we thus consider 2OMePS AONs currently the most favorable compounds, at least for targeted DMD exon 46 skipping.

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

“…This variability has been demonstrated in 2OMeAO-induced skipping of a variety of human exons in tissue culture and in a mouse carrying the human genomic dystrophin gene. But these studies have also found that a large proportion of exons are readily skipped by the use of appropriate AO sequences (21,22).…”

Comparative Analysis of Antisense Oligonucleotide Sequences for Targeted Skipping of Exon 51 During Dystrophin Pre-mRNA Splicing in Human Muscle

“…One strategy, based on previous results obtained in mouse dystrophin exon 23 (Graham et al, 2004), was that of designing an overlapping stepped array of 2-OMe AOs complementary to the 5Ј (donor) splice site of intron 51. The other strategies examined other potential splice motifs across exon 51, including the 3Ј (acceptor) splice site and the branch point, as well as exon-internal sequences (Aartsma-Rus et al, 2004b;Wilton et al, 2007). After initial characterization in each partner laboratory (Table 2) (Aartsma- Rus et al, 2002Rus et al, , 2005Harding et al, 2007), eight AOs, six targeting the 5Ј splice site designed in the first partner laboratory (RH) (C20 to H20; Fig.…”

In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy

“…the recent PNA-Pip series) [13,14] and, therefore, the availability of reliable, rapid in vitro screens to accelerate AO discovery would be highly advantageous. In vitro screens of AO splice correcting activity have been carried out widely using H 2 K mdx cells [18] and also using DMD patient-derived cells [19][20][21][22]; however, it has been reported that such in vitro systems work less well for neutrally charged AOs (e.g. PMO or PNA) and their derivatives [20,23], which can present transfection difficulties, and that the in vitro activity of different AO chemistries seldom correlates well with their in vivo efficacy in mdx mice.…”