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. 2019 Dec 12;20(24):6274.
doi: 10.3390/ijms20246274.

Ataluren for the Treatment of Usher Syndrome 2A Caused by Nonsense Mutations

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Free PMC article

Ataluren for the Treatment of Usher Syndrome 2A Caused by Nonsense Mutations

Ananya Samanta et al. Int J Mol Sci. .
Free PMC article

Abstract

The identification of genetic defects that underlie inherited retinal diseases (IRDs) paves the way for the development of therapeutic strategies. Nonsense mutations caused approximately 12% of all IRD cases, resulting in a premature termination codon (PTC). Therefore, an approach that targets nonsense mutations could be a promising pharmacogenetic strategy for the treatment of IRDs. Small molecules (translational read-through inducing drugs; TRIDs) have the potential to mediate the read-through of nonsense mutations by inducing expression of the full-length protein. We provide novel data on the read-through efficacy of Ataluren on a nonsense mutation in the Usher syndrome gene USH2A that causes deaf-blindness in humans. We demonstrate Ataluren´s efficacy in both transiently USH2AG3142*-transfected HEK293T cells and patient-derived fibroblasts by restoring USH2A protein expression. Furthermore, we observed enhanced ciliogenesis in patient-derived fibroblasts after treatment with TRIDs, thereby restoring a phenotype that is similar to that found in healthy donors. In light of recent findings, we validated Ataluren´s efficacy to induce read-through on a nonsense mutation in USH2A-related IRD. In line with published data, our findings support the use of patient-derived fibroblasts as a platform for the validation of preclinical therapies. The excellent biocompatibility combined with sustained read-through efficacy makes Ataluren an ideal TRID for treating nonsense mutations based IRDs.

Keywords: Ataluren; Retinitis pigmentosa; TRID; Usher syndrome; ocular therapy; patient-derived fibroblasts; translational read-through.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Translation: normal procedure, at a premature stop codon (PTC) and in the presence of translational read-through inducing drugs (TRIDs). (A) During elongation of the translation process, the binding of a cognate aminoacyl-tRNA (tRNA; grey rounded square) and the elongation factor eEF1A (grey rectangle) to the mRNA triplet at the A site catalyses the peptide bond formation between the nascent polypeptide (dark blue) at the P site and the new amino acid. At the site of a stop codon (red) a termination complex (eRF1, eRF3 GTP, eIF4E, eIF4G, PABPC1; orange) is formed. Upon formation of the termination complex, the release of the nascent polypeptide chain from the tRNA is induced. (B) In-frame nonsense mutations introduce a stop codon into the genomic sequence resulting in PTC in the mRNA. Translation of the mRNA stops (red X) resulting in a shortened polypeptide. This truncated polypeptide can have deleterious effects to the cells, including gain-of-function and loss-of function effects. (C) Translational read-through inducing drugs (TRIDs, green octagon) bind to ribosomes and can thereby enhance the translational read-through of PTCs. This results in the expression of full-length proteins. Resulting proteins might have an altered amino acid (yellow) at the site of the PTC.
Figure 2
Figure 2
Ataluren induced translational read-through of the USH2A_p.G3142* nonsense mutation in transiently USH2AG3142*-transfected HEK293T cells. (A) Scheme of wildtype USH2A isoform b protein. Extra: extracellular domain; EGF-LAM: laminin-type EGF (epidermal growth factor)-like modules; FN3: fibronectin type II motif; intra: intracellular domain; LamG: laminin G domain; LamGL: laminin G-like domain; SP: signal peptide; TM: transmembrane domain; star indicates a PDZ-binding motif (PBM). (B) Scheme of reporter construct of USH2A carrying the p.G3142* nonsense mutation (USH2A31G3142*) used in present study. The reporter construct contains the extracellular FN3 domains 18-24 and 35. The coding sequence is flanked by an HA-tag and Myc-tag, respectively. (C,D) HEK293T cells were transiently transfected with the wildtype (USH2A+) and mutated USH2A (USH2AG3142*) reporter constructs. Six h later USH2AG3142*-transfected cells were treated with DMSO (control) Gentamicin (Gent, 1 mg/ml) or Ataluren (10 µg/µl). (C) Co-immunolabelling applying anti-HA (red) and anti-Myc antibodies (green) validated the translational read-through of the nonsense mutation after Gentamicin and Ataluren treatment in transiently transfected USH2AG3142* HEK293T cells. Nuclei are stained with DAPI (blue). All images are in the same magnification, scale bar: 25 µm. (D) Western blot analysis with anti-Myc antibody detected full-length USH2A expression (∼98 kDa) after Gentamicin and Ataluren treatment of USH2AG3142*-transfected cells. Both TRIDs increases the expression of the mutated reporter construct (arrow head). Actin (42 kDa) was used as a loading control. (E) Quantification of the Western Blot analysis. Error bars represent the standard deviation, p < 0.001(***), p < 0.01 (**).
Figure 3
Figure 3
Ataluren induced translational read-through in patient-derived fibroblasts. (A) Fibroblasts of healthy donors, untreated USH2A patient-derived fibroblasts (untr.) and USH2AG3142* patient-derived fibroblasts treated with Gentamicin (Gent, 1mg/ml) or Ataluren (Ata., 5 µg/µL, 10 µg/µL), respectively, were subjected to Western blot analysis. A stronger USH2A expression was detected in fibroblasts of healthy donors and patient-derived cells treated with 5 µg/µL Ataluren compared the untreated and Gentamycin-treated cells. (B) Quantification of Western blot analysis demonstrated the significantly increased USH2A protein levels in 5 µg/µL Ataluren treated USH2A patient-derived fibroblasts compared to untreated patient-derived cells (* p < 0.05). Three independent experiments are included. (C) Indirect immunofluorescence analysis of healthy fibroblasts and USH2A patient-derived fibroblasts. Immunofluorescence staining revealed a membranous USH2A localisation in fibroblasts of a healthy donor and in Ataluren-treated USH2AG3142* patient-derived cells. A faint, punctuated USH2A protein expression was observed in untreated USH2A patient-derived cells. Treatment with Gentamicin partially restored USH2A localization at the membrane in patient-derived cells. Scale bar representative for the first three columns: 25 µm; scale bar representative for all zoom images: 5 µm. Three independent experiments were performed.
Figure 4
Figure 4
Ataluren increases the number of ciliated patient-derived fibroblasts. (A) Workflow. Healthy control human-derived fibroblasts and USH2AG3142* patient-derived fibroblasts were seeded. 24 h later culture media was changed to starvation media. Cells were treated with dimethyl sulfoxide (DMSO), Gentamicin (1 mg/mL), or Ataluren (5 µg/µL), respectively. After 24 h cells were subjected for immunofluorescence analysis and the number of ciliated cells was counted. Four independent experiments were included. (B) Anti-acetylated tubulin (Actub, green) was used as a ciliary marker, Pericentrin 2 (PCTN2, red) as basal body marker and DAPI to stain the nucleus. All images are in the same magnification, scale bar represents: 25 µm. (C) The graph represents the percentage of ciliated cells versus the total number of cells in healthy control (light grey) and in patient-derived fibroblasts (dark grey), respectively. A significant decrease in the percentage of ciliated cells was observed in untreated, DMSO- or Gentamicin-(Gent)-treated USH2AG3142* patient-derived cells versus healthy-donor derived fibroblasts. Ataluren treatment (5 µg/µl) restored the percentage of ciliated cells of patient-derived cells similar to that of healthy donors.

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