Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells

Abstract

Due to frame-shifting mutations in the DMD gene that cause dystrophin deficiency, Duchenne muscular dystrophy (DMD) patients suffer from lethal muscle degeneration. In contrast, mutations in the allelic Becker muscular dystrophy (BMD) do not disrupt the translational reading frame, resulting in a less severe phenotype. In this study, we explored a genetic therapy aimed at restoring the reading frame in muscle cells from DMD patients through targeted modulation of dystrophin pre-mRNA splicing. Considering that exon 45 is the single most frequently deleted exon in DMD, whereas exon (45+46) deletions cause only a mild form of BMD, we set up an antisense-based system to induce exon 46 skipping from the transcript in cultured myotubes of both mouse and human origin. In myotube cultures from two unrelated DMD patients carrying an exon 45 deletion, the induced skipping of exon 46 in only approximately 15% of the mRNA led to normal amounts of properly localized dystrophin in at least 75% of myotubes. Our results provide first evidence of highly effective restoration of dystrophin expression from the endogenous gene in DMD patient-derived muscle cells. This strategy may be applicable to not only >65% of DMD mutations, but also many other genetic diseases.

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

“…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]. Despite these and other in vitro studies, little agreement exists on the optimal parameters for in vitro AO screening, no studies to date have compared across different AO backbone chemistries and AO-peptide modifications, and little if any data exist on the value of in vitro screens as predictors of in vivo AO activity across a wide range of AO compounds.…”

“…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.…”

Heterogous Dystrophin mRNA Produced by a Novel Splice Acceptor Site Mutation in Intermediate Dystrophinopathy

“…Skipping of exon 19 in myocytes from a DMD patient with an exon 20 deletion led to the production of an in-frame dystrophin mRNA lacking both the exon 19 and 20 sequence, resulting in dystrophin-positive cells (36). In addition, it was shown that the skipping of exon 46 induced using antisense oligonucleotide complementary to a purine-rich sequence led to the production of dystrophin in DMD-derived muscle cells (37). At the same time, the induction of exon skipping with antisense oligonucleotides complementary to splicing consensus sequences was shown to lead to the production of an internally truncated dystrophin in the mdx mouse, an animal model of DMD (38).…”

Un saut symbolique mais encourageant dans le traitement de la myopathie de Duchenne

“…C'est l'équipe de G.J. van Ommen à Leiden (Pays-Bas) qui a proposé la première ce concept d'épisso-thérapie, et qui en a poussé les feux avec une remarquable constance et efficacité [9][10][11] ce qui a abouti à la réalisation du premier essai clinique par SET [1] que nous commentons ici. [12], mais pose le problème de l'immunogénicité du vecteur viral.…”

“…[12], mais pose le problème de l'immunogénicité du vecteur viral. L'efficacité de la stratégie du SET a été validée, tant ex-vivo (myoblastes en culture) qu' in vivo (par voie intramusculaire ou intravasculaire) sur des modèles animaux de dystrophinopathies dues à des mutations nulles (stop de la souris mdx ; mutation d'épissage du chien GRMD) [9][10][11][12][13][14][15]. Les résultats ont été spectaculaires puisqu'une réexpression stable de la « quasi-dystrophine » attendue a été obtenue avec un rendement très significatif.…”