Therapeutic exon skipping for dysferlinopathies?

Abstract

Antisense-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) currently tested in clinical trials. The aim is to reframe dystrophin transcripts using antisense oligonucleotides (AONs). These hide an exon from the splicing machinery to induce exon skipping, restoration of the reading frame and generation of internally deleted, but partially functional proteins. It thus relies on the characteristic of the dystrophin protein, which has essential N- and C-terminal domains, whereas the central rod domain is largely redundant. This approach may also be applicable to limb-girdle muscular dystrophy type 2B (LGMD2B), Myoshi myopathy (MM) and distal myopathy with anterior tibial onset (DMAT), which are caused by mutations in the dysferlin-encoding DYSF gene. Dysferlin has a function in repairing muscle membrane damage. Dysferlin contains calcium-dependent C2 lipid binding (C2) domains and an essential transmembrane domain. However, mildly affected patients in whom one or a large number of DYSF exons were missing have been described, suggesting that internally deleted dysferlin proteins can be functional. Thus, exon skipping might also be applicable as a LGMD2B, MM and DMAT therapy. In this study we have analyzed the dysferlin protein domains and DYSF mutations and have described what exons are promising targets with regard to applicability and feasibility. We also show that DYSF exon skipping seems to be as straightforward as DMD exon skipping, as AONs to induce efficient skipping of four DYSF exons were readily identified.

Figure 1

Dysferlin domains relative to DYSF exons. Dysferlin contains six or seven calcium-dependent C2 lipid binding domains (C2), a transmembrane domain (T), a ferl domain (L), FerA and FerB domains (A and B, respectively) and Dysf_N and Dysf_C domains (N and C, respectively). The C2 and transmembrane domains have a function in membrane repair. The function of other domains is yet unknown.

Figure 2

Antisense-mediated exon skipping. Left panel: in this example, a mutation within exon 32 results in a premature stop codon (indicated by the transition of black to white in the pre-mRNA (top) and mRNA (middle), which leads to a prematurely truncated protein (bottom). Right panel: when antisense oligonucleotides (AON) targeting exon 32 are used, they will hybridize to this exon, thus hiding it from the splicing machinery, resulting in the skipping of this exon. As exon 32 in in-frame (its length is divisible by three), skipping will not disrupt the reading frame (the mRNA becomes black in the middle panel) and a full-length protein, which misses a little bit in the middle, can be generated (bottom).

Figure 3

Dysferlin exons. In-frame exons are depicted in white, out-of-frame exons in black. Exons or combinations of exons can be skipped without disrupting the reading frame, when the resulting ends fit (eg, exons 39 and 40 can be skipped, as the end of exon 38 fits to the beginning of exon 41).

Figure 4

RT-PCR analysis of AON-treated control cell cultures. AONs 19–2, 24–1, 24–2, 30–1, 30–2 and 34–1 are effective, whereas 19–1, 32, 34–2 and C (a control AON targeting the DMD (dystrophin) gene) are not. Correct exon skipping was confirmed using sequence analysis (data not shown). No exon 19, 24, 32 or 34 skipping could be observed in nontreated (NT) cells, whereas for exon 30 low levels of physiological skipping were observed. AON treatment significantly increased these levels from <10 to >90%. Skipping percentages using Lab-on-a-chip (Agilent, Amstelveen, the Netherlands) are indicated below each skip. Note that the intensity of the skip products is lower, due to the smaller fragment length (our efficiency assessment corrects for this). –RT and H₂O are negative controls. M is size marker.