G418 as a suppression therapy for beta-thalassemia disease

Dias, Patrícia; Gomes-Duarte, Andreia; Romão, Luísa

http://hdl.handle.net/10400.18/5141

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Authors

Dias, Patrícia

Gomes-Duarte, Andreia

Romão, Luísa

Abstract(s)

Premature translation-termination codons (PTCs or nonsense codons) can arise from mutations in germ or somatic cells. The introduction of a PTC into an mRNA can trigger nonsense-mediated decay (NMD), an important mRNA surveillance mechanism that typically recognizes and degrades mRNAs containing PTCs to prevent the synthesis of C-terminally truncated proteins potentially toxic for the cell. The physiological relevance of NMD is manifested by the fact that about one third of genetic disease-associated mutations generate PTCs, including -thalassemia. In recent years, a novel therapeutic approach entitled suppression therapy has been developed based on low molecular weight compounds to induce the translation machinery to recode a PTC into a sense codon, the so called “readthrough” (or suppression). Here, by using a model of constructs containing the 5’ part of the normal, or nonsense-mutated, human -globin gene fused to the firefly luciferase gene as a reporter, we intend to prove the principle that the suppression therapy can restore enough -globin protein to outweight the manifestations of -thalassemia. Our results from bioluminescence assays and Western blot analyses have shown that the aminoglycoside G418 is able to suppress a nonsense mutation at codon 15 or 39 of the human -globin mRNA, in cultured HEK293 cells. We are now interested in stablishing how NMD inhibition can increase the efficiency of suppression therapy. A depper study on the suppression therapy is crucial, as it offers a major approach to treat a wide range of inherited pathologies.