Muscle dysfunction and structural defects of dystrophin‐nullsapjemutant zebrafish larvae are rescued by ataluren treatment

Abstract

Sapje zebrafish carry a mutation in the dystrophin gene, which results in a premature stop codon, and a severe muscle phenotype. They display several of the structural characteristics of Duchenne muscular dystrophy (DMD). Ataluren (PTC124) is proposed to cause readthrough of premature stop codons and has been introduced as a potential treatment of genetic disorders. Clinical trials in DMD have shown promise, although with complex dose dependency. We have established physiology techniques, enabling high resolution of contractile function in skeletal muscle of zebrafish larvae. We aimed to provide a mechanical analysis of sapje larval muscle and examine effects of ataluren. Homozygous 5 d postfertilization (dpf) sapje larvae exhibited structural defects with 50% decrease in active tension. Ataluren (0.1-1 μM, 3-5 dpf) improved contractile function (~60% improvement of force at 0.5 μM) and dystrophin expression. Controls were not affected. Higher doses (5 μM, 35 μM) impaired contractile function, an effect also observed in controls, suggesting unspecific negative effects at high concentrations. In summary, Sapje larvae exhibit impaired contractile performance and provide a relevant DMD model for functional studies. Ataluren significantly improves skeletal muscle function in the sapje larvae, most likely reflecting an observed increase in dystrophin expression. The bell-shaped dose dependence in sapje resembles that previously reported in clinical DMD studies.

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

“…The weak readthrough activity seen here with the HA-LUC (UAA20) -SF allele is atypical of that seen with UAA PTCs in other mRNAs (3,4,14) and most likely reflects the influence of codon context on readthrough (29). Ataluren's influence over the extent of specific tRNA selection implies that this drug's target could be the ribosome, a conclusion consistent with the drug's markedly diminished efficacy in the presence of tobramycin (Fig.…”

“…1 and SI Appendix, Fig. S3) (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)23); (ii) the effects on luciferase activity could not be independently replicated by others (24) or by us (SI Appendix , Fig. S3C); (iii) ataluren's putative luciferase inhibitory activity depends on a specific enzyme substrate (25); and (iv) most of the hypothetical inhibitory molecule, PTC124-AMP (22), is rapidly converted to the active readthrough molecule PTC124 (ataluren) under conditions of in vitro translation (SI Appendix, Fig.…”

“…Multiple experimental approaches with cell lines, patient cells, animal models, and genetic disorder patients have demonstrated that the drug ataluren can restore expression to genes and mRNAs otherwise inactive because of the presence of premature nonsense codons (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)23). Collectively, such experiments have strongly suggested that ataluren promotes nonsense suppression, i.e., the insertion of near-cognate tRNAs at PTCs, but direct evidence for this activity has been lacking.…”

“…To date, ataluren has been shown to restore function to more than 20 different disease-specific or reporter nonsense alleles in systems ranging in complexity from in vitro translation to cell culture to mouse models and human patients (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). In the latter, Translarna clinical trials have demonstrated restoration of full-length functional protein in Duchenne muscular dystrophy and cystic fibrosis nonsense mutation patients (10,11,13,15,16).…”

Ataluren: First Global Approval

“…The data obtained showed that ataluren allowed dystrophin to be made in cells in which it was previously absent, to be delivered to the proper cellular location, and to induce restoration of muscle function [18]. In a zebrafish model of DMD, improvements in skeletal muscule contractile function were observed at ataluren concentrations of 0.1-1 lM, but muscle contraction was impaired at higher concentrations (5 lM) [19]. The results of a phase IIa study conducted in children with DMD (NCT00264888) confirmed that ataluren increased dystrophin expression, with significant increases from baseline documented during treatment [20].…”