Antisense oligo-mediated multiple exon skipping in a dog model of duchenne muscular dystrophy

Abstract

Exon skipping is currently one of the most promising molecular therapies for Duchenne muscular -dystrophy (DMD). We have recently developed multiple exon skipping targeting exons 6 and 8 in -dystrophin mRNA of canine X-linked muscular dystrophy (CXMD), an animal model of DMD, which exhibits severe dystrophic phenotype in skeletal muscles and cardiac muscle. We have induced efficient exon skipping both in vitro and in vivo by using cocktail antisense 2'O-methyl oligonucleotides (2'OMePS) and cocktail phosphorodiamidate morpholino oligomers (morpholinos, or PMOs) and ameliorated phenotype of dystrophic dogs by systemic injections. The multiple exon skipping (double exon skipping) shown here provides the prospect of choosing deletions that optimize the functionality of the truncated dystrophin protein for DMD patients by using a common cocktail that could be validated as a single drug and also potentially applicable for more than 90% of DMD patients.

Fig. 1

Multiple exon skipping in dystrophic dogs. (a) Schematic outline of the protocol. A splice site mutation in intron 6 leads to deletion of exon 7 at mRNA level in dystrophic dogs. To restore the reading frame, two additional exons (exon 6 and exon 8) need to be skipped (removed) by three oligo cocktail of antisense. Exon 9 is known as alternative splice site. (b) RT-PCR and cDNA sequencing after exon skipping in dystrophic dogs. Left panel; RT-PCR reveals exon 6–9 skipped in-frame products (101 bp) in dystrophic dogs after the treatment of cocktail oligos. Alternative splice site Exon 9 is also mostly removed from the resulting mRNA. Right panel; Exon-skipping patterns are further confirmed by cDNA sequencing.

Fig. 2

Frozen muscle samples from dystrophic dogs.

Fig. 3

Recovery of dystrophin expression after cocktail antisense transfection in dystrophic dog myoblast. Dystrophin expression and DAPI nuclear double staining of wild-type myotubes (a), cocktail of Ex6A, Ex6B, and Ex8A 2’OMePS transfected myotubes (b), and nontreated CXMD myotubes (c). Dystrophin C-terminal antibody DYS-2 is used. Bar: 50 µm.

Fig. 4

Recovery of dystrophin expression after 7 × 200 mg/kg intravenous cocktail morpholino injections. Dystrophin expression of wild-type dog muscle (a), cocktail of Ex6A, Ex6B, and Ex8A PMOs injected dog muscle (b), nontreated CXMD dogs (c), Western blotting analysis with dystrophin antibody (d). Bar: 100 µm.