Strategies for the adult haemoglobin (HbA) production in β0-thalassemia patients

Background: 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. Currently, there are two approaches to directly overcome diseases caused by nonsense mutations: gene therapy, meaning introduction of an exogenous gene, and translational read-through induced by aminoglycosides, which decrease the accuracy of translation elongation and reduce the efficacy of the translation termination machinery. 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 codons. On the other hand, in recent years, many investigators have been studying the development of safe lentiviral vectors to avoid the side effects as activation of oncogenes and transcription silencing. Aim: Our purpose was to verify the effects and clinical utility in b0- thalassemia of translational read-through and gene therapy. Methods: First, we started the development of a cellular model of the b039- thalassemia mutation that could be used for the screening of high numbers of aminoglycosides and analogous molecules. We produced two lentiviral vectors containing the bwt- or b039-thalassemia globin gene under a minimal LCR control region and used such constructs for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different copynumber of the integrated constructs. These clones were then treated with geneticin (G418) and other aminoglycosides and the production of β-globin was analysed by FACS analysis. Secondly, we screened several thalassemia patients and isolated erythroid progenitors from such individuals resulted homozygous for the b039 mutation. Then, we treated the cells with geneticin and analysed the production of b-globin and adult haemoglobin (HbA) by FACS and HPLC analysis, respectively. Finally, we subjected the b039 erythroid precursor cells to gene therapy, through the use of the lentiviral vector expressing the βwt-globin mRNA, with the aim to verify the increase of HbA through HPLC analysis. Moreover, by treating the transduced cells with mithramycin we wanted to verify the combined effects of gene therapy and fetal haemoglobin (HbF) induction. Main results: We obtained and characterized six clones carrying the b039- globin gene and seven clones carrying the bwt-globin gene, to be used as a control. We then chose two clones, a bwt and a b039, showing similar content of mRNA and used them to test various aminoglycosides to verify their read-through activity. Geneticin (G418) and gentamicin showed the highest capacity to readthrough of nonsense mutation, although the effect of G418 was found to be higher than that displayed by gentamicin. A significant increase in β-globin containing cells was also detected when b039 progenitor cells were treated with G418. This last result was confirmed by HPLC analysis, which showed an increase in the relative concentration of HbA compared to total Hbs. Finally, we obtained a high increase in the proportion of HbA when the same progenitor cells were subjected to gene therapy, through transduction with the lentiviral vector containing the bwt-globin gene, and a clear increment of HbF was detected after treatment with mithramycin. Conclusion: Both the therapeutic approaches for the cure of β0-thalassemias analysed in this work, namely translational read-through and gene therapy, while still presenting many disadvantages, including toxicity of aminoglycosides and the uncertainty of the effects of lentiviral vectors on endogenous genes, can be considered techniques with a high curative potential. Naturally, in the case of translational read-through is hoped the identification of new compounds with a therapeutic effect similar to but greater than that of aminoglycosides, with less toxicity and oral bioavailability, while in the case of gene therapy, further investigations should be made to eliminate its well known disadvantages, primarily including the possible activation of oncogenes.