Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides

Abstract

Deletions and point mutations in the gene encoding the cytoskeletal protein dystrophin and its isoforms cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy (BMD), largely depending on whether the reading frame is lost or maintained respectively. Frameshift mutations tend to result in a lack of dystrophin at the sarcolemma, destabilization of the membrane and degeneration of skeletal muscle. The mdx mouse is a valuable animal model of DMD as it bears a nonsense point mutation in exon 23 of the murine DMD gene leading to an absence of dystrophin expression in the muscle sarcolemma and muscular dystrophy. This report represents a novel approach to correct dystrophin deficiency at the post-transcriptional level by transfection of muscle cells with antisense RNA. Essentially, 2'- O -methyl oligoribonucleotides (2'OMeRNA) were delivered to the nuclei of primary mdx myoblasts in culture. Dystrophin expression was observed in the sarcolemma of transfected mdx myotubes after transfection by an oligonucleotide complementary to the 3' splice site of murine dystrophin intron 22. Direct sequencing of RT-PCR products from these cells revealed precise splicing of exon 22 to exon 30, skipping the mutant exon and creating a novel in-frame dystrophin transcript. As patients with comparable in-frame internal deletions show relatively mild myopathic symptoms, this may in the future offer a therapeutic approach for DMD, as well as for other inherited disorders.

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

“…Thus, the in vitro system and the conditions reported in the present study will be valuable for the rapid screening of novel splice correcting AOs, including novel AO chemistries [8] and AO-peptide conjugates [13], and should accelerate future therapeutic advances for DMD. The earliest demonstrations of successful AO-induced exon skipping of the dystrophin were demonstrated in vitro in human lymphoblastoid and mdx mouse muscle cells [18,21], skipping DMD exons 19 and exons 22-30, respectively. Subsequently, numerous in vitro exon skipping studies have been undertaken demonstrating precise skipping of DMD exon 23 in mdx mouse muscle cells [29] with protein correction in mdx mice in vivo [6]; successful DMD exon 46 skipping in patient-derived muscle cells [22]; further mdx exon 23 2 OMePS sequence refinement and evaluation of AO concentrations [30]; evaluation of 'leashed' PMO AOs [23]; the comparative effects of different 2 OMePS, PMO, PNA and LNA AOs for skipping DMD exon 46 in human muscle cells [2]; the influence of AO length on AO splice correcting efficacy [31]; optimization and selection of a human DMD exon 51 skipping AO for clinical trial [32]; and the exon skipping activity of PNA and PNApeptide AOs in mdx muscle cells [8,13,14].…”

“…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.…”

Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product

“…Alternatively, correction of the endogenous dystrophin gene was attempted using chimeric RNA/DNA oligonucleotides 10,11 or antisense 2'-O-methyl oligoribonucleotides. 12 The antibiotic gentamicin has been reported to increase read-through dystrophin protein over a premature stop codon, 13 and clinical trials have been conducted recently. 14 On the other hand, compensation for dystrophin lack by up-regulation of endogenous utrophin 15 is another possible strategy.…”

Le saut d’exon thérapeutique : un espoir pour les dystrophinopathies

“…Goyenvalle [5,6]. Mais ni l'efficacité, ni la stabilité n'atteignaient un niveau de significativité clinique.…”

“…Dès 1991, M. Matsuo [3] avait insisté sur l'importance des phénomènes d'épis-sage dans le déterminisme et la théra-peutique des dystrophinopathies. Plus récemment, d'autres groupes se sont engagés dans la voie de l'épissothéra-pie, comme alternative à la thérapie génique classique qui n'a pas encore remporté les succès escomptés [4][5][6]. Une restauration de la synthèse de dystrophine musculaire chez la souris mdx par exclusion post-transcriptionnelle de l'exon porteur de la mutation (stop sur l'exon 23) a été rapportée par plusieurs…”