Ataluren as an agent for therapeutic nonsense suppression

Abstract

The interplay of translation and mRNA turnover has helped unveil how the regulation of gene expression is a continuum in which events that occur during the birth of a transcript in the nucleus can have profound effects on subsequent steps in the cytoplasm. Exemplifying this continuum is nonsense-mediated mRNA decay (NMD), the process wherein a premature stop codon affects both translation and mRNA decay. Studies of NMD helped lead us to the therapeutic concept of treating a subset of patients suffering from multiple genetic disorders due to nonsense mutations with a single small-molecule drug that modulates the translation termination process at a premature nonsense codon. Here we review both translation termination and NMD, and our subsequent efforts over the past 15 years that led to the identification, characterization, and clinical testing of ataluren, a new therapeutic with the potential to treat a broad range of genetic disorders due to nonsense mutations.

Figure 1. Ataluren treatment promotes dystrophin and CFTR synthesis in mouse models of nmDMD and nmCF

a, Premature translation termination. The figure depicts a closed-loop mRNP in the process of being translated. Translational elongation doesn’t extend to the normal termination codon (red stop sign), but is instead halted by the premature termination codon (yellow stop sign), leading to the accumulation of a truncated polypeptide. b, Ataluren: 3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid (PTC124^™) (71). c, Ataluren treatment of mdx mice rescues the dystrophic phenotype of affected muscles. Immunohistochemistry of indicated muscle cross-sections to visualize dystrophin in the tibialis anterior, diaphragm, and heart. The white arrow in the tibialis anterior negative control designates a revertant fibre. From (71). d, hCFTR expression in submucosal glands of intestinal tissues from Cftr−/− hCFTR-G542X mice treated with ataluren (PTC124). Samples from the duodenum of cftr −/− hCFTR G542X mice treated with 0.3 or 0.9 mg/ml ataluren in the Peptamen liquid diet. Samples were incubated with either preimmune or hCFTR-NBD1 serum as indicated. After incubation with a fluorescent secondary antibody, the samples were visualized by fluorescence microscopy. From (72).

Figure 2. Ataluren-mediated dysferlin and CFTR expression

a, Treatment of Miyoshi myopathy patient myotubes with ataluren yields ~15% of the levels of dysferlin present in unaffected myotubes. Comparisons employed western blotting and densitometry, and normalization to the levels of α-sarcoglycan in unaffected and Miyoshi myotubes. b, Dysferlin expression in myotubes from an unaffected human and from a Miyoshi patient. The Miyoshi myotubes do not express dysferlin unless infected with recombinant adenovirus that expresses full-length human dysferlin (Ad-DYSF) or treated with ataluren. Myotubes stained with either anti-myosin (red) or anti-dysferlin (green) antibodies. All scale bars represent 20 μm. From (82). c, Ataluren-mediated increase in expression of nasal epithelial cystic fibrosis transmembrane conductance regulator in a phase 2a CF patient. An immunofluorescence assay demonstrates that a patient from a phase 2a trial of ataluren efficacy in nmCF manifests full-length nasal epithelial cystic fibrosis transmembrane conductance regulator protein (green immunofluorescence) at the end of the trial (B), but not at its onset (A). Paired pre-and end-of-treatment immunostaining images are from a 17-year-old female patient with nmCF. She received ataluren 4, 4, 8 mg/kg for 14 days followed by 14 days off treatment, then received ataluren 10, 10, 20 mg/kg for 14 days. From (96). d, Restoration of chloride transport in phase 2a CF patients treated with ataluren. Seventeen nmCF patients were treated with ataluren (4, 4, and 8 or 10, 10, and 20 mgxkg⁻¹) for 84 days, and followed up without drug for 28 days. Total chloride transport (mv) was monitored throughout the trial. Data are presented as mean±sem. p-values from paired t-test comparing on-study time point versus corresponding baseline. From (97).

Figure 3. Ataluren manifests a bell-shaped dose response

Myotubes from a phase 2a nmDMD patient were cultured in the presence of different concentrations of ataluren and assayed for the accumulation of dystrophin and a control protein by immunofluorescence assays.