Correction of ClC-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy

Abstract

In myotonic dystrophy (dystrophia myotonica [DM]), an increase in the excitability of skeletal muscle leads to repetitive action potentials, stiffness, and delayed relaxation. This constellation of features, collectively known as myotonia, is associated with abnormal alternative splicing of the muscle-specific chloride channel (ClC-1) and reduced conductance of chloride ions in the sarcolemma. However, the mechanistic basis of the chloride channelopathy and its relationship to the development of myotonia are uncertain. Here we show that a morpholino antisense oligonucleotide (AON) targeting the 3′ splice site of ClC-1 exon 7a reversed the defect of ClC-1 alternative splicing in 2 mouse models of DM. By repressing the inclusion of this exon, the AON restored the full-length reading frame in ClC-1 mRNA, upregulated the level of ClC-1 mRNA, increased the expression of ClC-1 protein in the surface membrane, normalized muscle ClC-1 current density and deactivation kinetics, and eliminated myotonic discharges. These observations indicate that the myotonia and chloride channelopathy observed in DM both result from abnormal alternative splicing of ClC-1 and that antisense-induced exon skipping offers a powerful method for correcting alternative splicing defects in DM. IntroductionDystrophia myotonica (myotonic dystrophy) type 1 (DM1), the most common muscular dystrophy affecting adults, is caused by expansion of a CTG repeat in the 3′ untranslated region of the gene encoding the DM protein kinase (DMPK) (1). Evidence suggests that DM1 is not caused by abnormal expression of DMPK protein, but rather that it involves a toxic gain of function by mutant DMPK transcripts that contain an expanded CUG repeat (CUG exp ) (reviewed in ref.2). The transcripts containing a CUG exp tract elicit abnormal regulation of alternative splicing, or spliceopathy (3). The splicing defect, which selectively affects a specific group of pre-mRNAs, is thought to result from reduced activity of splicing factors in the muscleblind (MBNL) family (4), increased levels of CUG-binding protein 1 (3, 5), or both. Decreased activity of MBNL proteins can be attributed to sequestration of these proteins in nuclear foci of CUG exp RNA (6, 7).Previously we showed that transgenic mice expressing CUG exp RNA (human skeletal actin long repeat [HSA LR ] mice) displayed myotonia and chloride channel 1 (ClC-1) splicing defects similar to those observed in DM1 (8). We postulate that myotonia in the HSA LR model results from abnormal inclusion of exon 7a in the ClC-1 mRNA, owing to sequestration of MBNL1, a factor required for repression of exon 7a splicing in muscle fibers (4). This mechanism is supported by several lines of evidence: (a) inclusion of exon 7a causes frame shift and introduction of a premature termination codon in the ClC-1 mRNA (5, 8); (b) trun-

RNA repair restores hemoglobin expression in IVS2–654 thalassemic mice

“…Recent reports showed in vivo restoration of chloride channel function in myotonic dystrophy (21), factor VIII in a model of hemophilia (22), and dystrophin in the mdx mouse model of Duchenne muscular dystrophy (23)(24)(25) by oligonucleotideinduced modulation of splicing, an approach discovered in the Kole laboratory (6). These results, together with those reported here, indicate that the splicing-mediated pre-mRNA repair is not limited to a single disease.…”

CAG repeat RNA as an auxiliary toxic agent in polyglutamine disorders

“…19,37 Its pathomechanism was the first among neuromuscular disorders to be linked to aberrant functioning of mutant mRNA. The prevailing paradigm is that DM1 is a toxic RNA gain-of-function disease mediated by the expression of mutant (CUG) n expansion, [38][39][40] and its transcription appears to be both necessary and sufficient to cause disease. 18,20 Mutant RNA becomes trapped in the nucleus where it forms insoluble foci 19,41 that interfere with RNA splicing by altering functional levels of RNA-binding proteins such as the muscleblind-like family and CUG-binding protein 1 (CUGBP1).…”

“…For example, myotonia was proved to be caused by chloride channel (CLCN1) mis-regulation and inappropriate expression of fetal isoforms of the gene in DM1 adult tissues. 39 On the other hand, insulin…”

Myotonic Dystrophy: Therapeutic Strategies for the Future

“…Recently this approach was tested in two mouse models of myotonic dystrophy: an antisense oligonucleotide (AON) was designed to skip the abnormally included exon of the muscle chloride channel, exon 7a. 47 By suppressing inclusion of exon 7a, the AON restored the reading frame and produced a normal ClC-1 mRNA. The end result is production of a full-length, fully functional chloride channel and elimination of myotonia for up to 8 weeks after a single treatment.…”