Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice

Abstract

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, leading to the absence of the dystrophin protein in striated muscle. A significant number of these mutations are premature stop codons. On the basis of the observation that aminoglycoside treatment can suppress stop codons in cultured cells, we tested the effect of gentamicin on cultured muscle cells from the mdx mouse -an animal model for DMD that possesses a premature stop codon in the dystrophin gene. Exposure of mdx myotubes to gentamicin led to the expression and localization of dystrophin to the cell membrane. We then evaluated the effects of differing dosages of gentamicin on expression and functional protection of the muscles of mdx mice. We identified a treatment regimen that resulted in the presence of dystrophin in the cell membrane in all striated muscles examined and that provided functional protection against muscular injury. To our knowledge, our results are the first to demonstrate that aminoglycosides can suppress stop codons not only in vitro but also in vivo. Furthermore, these results raise the possibility of a novel treatment regimen for muscular dystrophy and other diseases caused by premature stop codon mutations. This treatment could prove effective in up to 15% of patients with DMD.

Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment

“…Recently, the possibly most encouraging therapeutic approach for a subset of muscular dystrophies has emerged with the report by Barton-Davis et al, 6 showing restoration of dystrophin levels to 10-20% of those normally found in the skeletal muscle of mdx mice, following subcutaneous injections of gentamicin, an aminoglycoside long known for its suppression of premature termination codons. Aminoglycoside antibiotics are active in the codonanticodon recognition of aminoacyl tRNAs during translation.…”

“…27,28 We chose to use the same antibiotic dose (34 mg/kg) as that in previously reported in vivo studies, since it showed no toxic effects. 6 Preliminary results showed that a significant readthrough level was obtained in murine skeletal muscle (4%), and that gentamicin increased this 1.5-fold. We then constructed a pCRFL reporter vector harboring the 319d mutation (pCRFL319d), which exhibited the highest gentamicin-induced readthrough efficiency and the highest induction factor (0.06-2.65%) in cultured cells assays (Table 1).…”

“…5,15 Secondly, the re-expression of a mutated gene after gentamicin treatment has been assessed in animal models, such as Mdx mdx or CFTR mutant mice. 6,8 However, the correlation between readthrough levels obtained in vitro or ex vivo and the re-expression observed in vivo remained uncertain, since several processes might modulate the primary molecular effect of the antibiotic on readthrough, in particular mRNA stabilization and protein accumulation.…”

“…4,5 The strength of this approach was first demonstrated in vivo by the work of Barton-Davis and co-workers, who demonstrated that gentamicin could partially restore expression of fulllength dystrophin in mdx mice with the X-linked muscular dystrophy mutation (Mdm mdx ), a C-T transition at position 3185, resulting in a termination codon replacing a glutamine codon in the dystrophin gene. 6 Negamycin, a dipeptide antibiotic with similar stop codon-misreading activity, has also been shown to restore dystrophin synthesis in the mdx mouse skeletal muscle. 7 More recently, Bedwell and co-workers demonstrated that gentamicin is effective in a transgenic mouse model of cystic fibrosis.…”

Long-term expression of full-length human dystrophin in transgenic mdx mice expressing internally deleted human dystrophins

“…6 There are many strategies for the induction of de novo dystrophin expression as a therapeutic approach for the treatment of DMD. These include systemic drug delivery, 7 gene modification at DNA and RNA levels, [8][9][10][11] cell transplantation [12][13][14] and gene delivery with a wide range of both viral [15][16][17] and nonviral [18][19][20] delivery systems. However, therapeutic levels, together with a stable longterm presence of dystrophin at the sarcolemma of the muscle fibres, are essential.…”