Antisense-mediated exon skipping: a versatile tool with therapeutic and research applications

Abstract

Antisense-mediated modulation of splicing is one of the few fields where antisense oligonucleotides (AONs) have been able to live up to their expectations. In this approach, AONs are implemented to restore cryptic splicing, to change levels of alternatively spliced genes, or, in case of Duchenne muscular dystrophy (DMD), to skip an exon in order to restore a disrupted reading frame. The latter allows the generation of internally deleted, but largely functional, dystrophin proteins and would convert a severe DMD into a milder Becker muscular dystrophy phenotype. In fact, exon skipping is currently one of the most promising therapeutic tools for DMD, and a successful first-in-man trial has recently been completed. In this review the applicability of exon skipping for DMD and other diseases is described. For DMD AONs have been designed for numerous exons, which has given us insight into their mode of action, splicing in general, and splicing of the DMD gene in particular. In addition, retrospective analysis resulted in guidelines for AON design for DMD and most likely other genes as well. This knowledge allows us to optimize therapeutic exon skipping, but also opens up a range of other applications for the exon skipping approach.

FIGURE 1.

Antisense-mediated exon skipping for Duchenne muscular dystrophy. The dystrophin protein (upper panel) contains an N-terminal actin-binding domain connected to a β-dystroglycan binding domain by the central rod domain. Dystroglycan is a transmembrane protein that is bound to the extracellular protein laminin-2. Dystrophin thus fulfills a bridge function in muscle fibers by linking the cytoskeletal actin to the extracellular matrix. In Duchenne muscular dystrophy (middle panel), the open reading frame is disrupted (in this example by a deletion of exons 48–50, the most common mutation in DMD patients), resulting in a premature stop codon and a truncated dystrophin, which is unable to fulfill its bridge function. Antisense oligoribonucleotides (AONs) can be employed to restore the open reading frame (lower panel). Specific AONs hybridize to exon 51 and hide this exon from the splicing machinery, resulting in the splicing of exon 51 with its flanking intron. This restores the open reading frame, allowing the generation of an internally deleted dystrophin, that contains both the actin- and dystroglycan binding domains and therefore is partially to largely functional.

FIGURE 2.

Box plots of the different groups of exons for the predicted donor and acceptor splice sites, exon length, and the lengths of the upstream, downstream, or flanking introns. Exon skippability is based on the report published by Wilton and colleagues (Wilton et al. 2007). The median value is indicated by a broad vertical line which is located within a box that contains all values between the 25th and 75th percentiles. The outer ranges are depicted by dotted lines and bordered by small horizontal lines. Outlying values are indicated by small circles. Splice site values were calculated with the Berkeley Drosophila Genome Project software for human splice site prediction. The predicted acceptor splice sites were significantly higher for poor skippable exons as calculated with the Kruskal–Wallis signed rank sum test (P-value 0.04, indicated with an asterisk). No significant differences were observed for the other parameters, although there is a trend for predicted donor splice sites to be somewhat lower for highly skippable exons (P-value 0.2).

FIGURE 3.

Antisense-mediated exon 8 skipping. Using AONs targeting exon 8 only the skipping of both exons 8 and 9 is observed. A likely explanation is that splicing of the downstream intron 8 (1.1 kb) precedes splicing of intron 7 (110 kb) and that the AONs do not affect the donor splice site of exon 8 (effective AONs used so far target either the acceptor splice site or the 5′ region of the exon). Thus, intron 8 can be spliced out and exons 8 and 9 are joined. As exon 8 AONs do disrupt the acceptor splice site of exon 8, the splicing machinery uses the first available acceptor splice site, which is that of exon 10 (because exons 8 and 9 are already joined).