Nonsense mutations that result in premature stop codons in the HBB gene cause β-thalassemia. This disease is characterized by a reduced hemoglobin level due to the lack of β-globin. Compounds that induce translational readthrough across the thalassemia-causing premature stop codon will have therapeutic benefits. Currently available molecules that induce translational readthrough lack specificity, and some of them show toxicity after prolonged use. In this study, we have developed an oligonucleotide-based approach to induce translational readthrough across the thalassemia-causing premature stop codon. Oligonucleotides that target HBB mRNA downstream of the premature stop codon could induce translational readthrough, generating a full-length β-globin protein. We show this effect using fluorescence- and luminescence-based readthrough assays and by Western blot. Remarkably, the amount of oligonucleotide-induced translational readthrough product is comparable to that of the protein generated by normal translation when there was no premature stop codon. Thus, these oligonucleotides, with certain modifications, have the potential to be used as drugs for the treatment of β-thalassemia. Also, this strategy can be extended to treat other genetic diseases caused by premature stop codons.

It is generally believed that human mature erythrocytes do not possess functional ribosomes, and therefore cannot synthesize proteins. However, the absence of translation is not consistent with the long lifespan of mature erythrocytes. They stay viable and functional for about 115 days in the circulatory system. Here, using highly pure preparation of human mature erythrocytes, we demonstrate the presence of translation by polysome profiling, [35S]methionine labelling and RiboPuromycylation. [35S]methionine labelling revealed that the translation in mature erythrocytes is about 10% of that observed in reticulocytes. We could observe polysomes by transmission electron microscopy in these cells. RNA-seq and quantitative RT-PCR performed on polysome fraction of these cells revealed that HBA (alpha globin) and HBB (beta globin) transcripts are translated. Using luciferase-based reporter assay and mutational studies, we show that the sequence of the 5' untranslated region is crucial for the translation of these transcripts. Furthermore, mature erythrocytes showed reduced expression of globin proteins (alpha and beta) when treated with translation inhibitors. Overall, we provide multiple lines of evidence for translation of globin mRNAs in human mature erythrocytes.

Stop codon readthrough (SCR) is the process where translation continues beyond a stop codon on an mRNA. Here, we describe a strategy to enhance or induce SCR in a transcript-selective manner using CRISPR-dCas13 system. Using specific guide RNAs, we targeted dCas13 to the downstream region of the canonical stop codons of mammalian AGO1 and VEGFA, which are known to exhibit natural SCR. Results of readthrough assays revealed the enhancement of SCR of these mRNAs (both exogenous and endogenous) caused by dCas13. This effect was associated with ribosomal pausing, which has been reported in several SCR events. Furthermore, our results show that CRISPR-dCas13 can induce SCR across premature termination codons (PTC) in the mRNAs of green fluorescent protein and TP53. Finally, we demonstrate the utility of this strategy in the induction of readthrough across the thalassemia-causing PTC in HBB mRNA. Thus, CRISPR-dCas13 can be programmed to enhance or induce SCR in a transcript-selective and stop codon-specific manner.