The coding sequence is flanked by an HA-tag and Myc-tag, respectively. The reporter construct contains the extracellular FN3 domains 18-24 and 35. ( B) Scheme of reporter construct of USH2A carrying the p.G3142* nonsense mutation (USH2A31 G3142*) used in present study. 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). ( A) Scheme of wildtype USH2A isoform b protein. Resulting proteins might have an altered amino acid (yellow) at the site of the PTC.Ītaluren induced translational read-through of the USH2A_p.G3142* nonsense mutation in transiently USH2A G3142*-transfected HEK293T cells. This results in the expression of full-length proteins. ( C) Translational read-through inducing drugs (TRIDs, green octagon) bind to ribosomes and can thereby enhance the translational read-through of PTCs. This truncated polypeptide can have deleterious effects to the cells, including gain-of-function and loss-of function effects. Translation of the mRNA stops (red X) resulting in a shortened polypeptide. ( B) In-frame nonsense mutations introduce a stop codon into the genomic sequence resulting in PTC in the mRNA. Upon formation of the termination complex, the release of the nascent polypeptide chain from the tRNA is induced. At the site of a stop codon (red) a termination complex (eRF1, eRF3 GTP, eIF4E, eIF4G, PABPC1 orange) is formed. ( 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. Translation: normal procedure, at a premature stop codon (PTC) and in the presence of translational read-through inducing drugs (TRIDs). The excellent biocompatibility combined with sustained read-through efficacy makes Ataluren an ideal TRID for treating nonsense mutations based IRDs.Ītaluren Retinitis pigmentosa TRID Usher syndrome ocular therapy patient-derived fibroblasts translational read-through. In line with published data, our findings support the use of patient-derived fibroblasts as a platform for the validation of preclinical therapies. In light of recent findings, we validated Ataluren´s efficacy to induce read-through on a nonsense mutation in USH2A-related IRD. 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. We demonstrate Ataluren´s efficacy in both transiently USH2A G3142*-transfected HEK293T cells and patient-derived fibroblasts by restoring USH2A protein expression. 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. 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. Therefore, an approach that targets nonsense mutations could be a promising pharmacogenetic strategy for the treatment of IRDs. Nonsense mutations caused approximately 12% of all IRD cases, resulting in a premature termination codon (PTC). The identification of genetic defects that underlie inherited retinal diseases (IRDs) paves the way for the development of therapeutic strategies.
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