Development of K562 cell clones expressing β‐globin mRNA carrying the β⁰39 thalassaemia mutation for the screening of correctors of stop‐codon mutations

Abstract

Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30 % of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in β039-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). 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 the premature termination codons. 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. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of β039-thalassaemia. In this context, we started the development of a cellular model of the β039-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the β039-thalassaemia globin gene under a minimal LCR (locus control region) 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 (also known as G418) and other aminoglycosides and the production of β-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the β039-globin mutation causing β-thalassaemia.

Production of β‐globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous β039 thalassemia patients

“…To test the effects of G418 on erythroid cells mimicking b 0 39-thalassemia, K562 cell clones carrying multiple copies of the normal and b 0 39-globin gene were used. The production of the lentiviral vectors used for the generation of the K562 cells clones carrying either bwt or b 0 39-globin genes has been reported elsewhere [43]. Briefly, the original 13,824 bp construct (pCCL.bwt.PGW) displays two LTR sequences, the SV40 origin of replication, a GFP gene under the control of the PGK promoter, the b-globin gene under the control of the b-globin gene promoter, and a minimal LCR of the human b-like globin gene cluster (see the map shown in Fig.…”

“…These vectors were transfected to K562 cells and several K562 cell clones isolated, expressing either the normal b-globin or the b 0 39-thalassemia globin genes at different levels. This system was proved to be suitable to detect readthrough activity [43].…”

“…In a recent article, we have described the development of a novel experimental system suitable to screen potential modifiers of biological consequences of stop mutations [43]. We have generated two lentiviral constructs, one containing the human normal b-globin gene and the other containing the b 0 39-thalassemia globin gene, both under the control of the b-globin promoter and a LCR cassette (see Fig.…”

Future alternative therapies for β-thalassemia

“…These findings have introduced new hopes for the development of a pharmacologic approach to treat thalassemic patients carrying the β0–39 mutation, which introduces a PTC in codon 39 of the β-globin gene and is one of the most frequent thalassemic mutations in the Mediterranean littoral [1]. To test whether aminoglycosides and analogous molecules can restore β-globin protein synthesis, human erythroid cells (K562) carrying a lentiviral construct containing the β0–39 globin gene were produced [126]. Treatment of these clones with geneticin (G418) and other aminoglycosides restored the production of β-globin [126].…”

“…To test whether aminoglycosides and analogous molecules can restore β-globin protein synthesis, human erythroid cells (K562) carrying a lentiviral construct containing the β0–39 globin gene were produced [126]. Treatment of these clones with geneticin (G418) and other aminoglycosides restored the production of β-globin [126]. More importantly, after fluorescence-activated cell sorting and high-performance liquid chromatography (HPLC) analyses, G418 was also demonstrated to correct the biological function of the β0–39 globin mRNA in erythroid precursor cells from β0–39 homozygous β-thalassemia patients [127].…”