Development of K562 cell clones expressing beta-globin mRNA carrying the beta039 thalassemia mutation for the screening of correctors of stop codon mutations

Nonsense mutations, giving rise to UAA, UGA, UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of about 30% of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy, thalassemia. For instance, in the beta039-thalassemia the CAG (Gln) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well described nonsense-mediated mRNA decay (NMD). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon-anticodon base pairing, inducing a ribosomal read through of premature termination codon. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read through of premature but not normal termination codon. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of beta039-thalassemia. In this context, we started the development of a cellular model of the beta039-thalassemia mutation that could be used for the screening of high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the beta039-thalassemia globin gene under a minimal LCR control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with geneticin (G418) and other aminoglycosides and the production of beta-globin was analyzed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the beta039-globin mutation causing beta-thalassemia.