Using 5′-PTMs to repair mutant β-globin transcripts

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Abstract
Trans-splicing has been used to repair mutant RNA transcripts via competition for the spliceosome using pre-trans-splicing molecules, or “PTMs.” Previous studies have demonstrated that functional PTMs can be designed for either 3′- or 5′-exon replacement, with a vast majority of the work to date focusing on repair of mutations within internal exons and via 3′-exon replacement. Here, we describe the first use oftrans-splicing to target the first exon and intron of a therapeutically relevant gene and repair the mutant RNA by 5′-exon replacement. Our results show that 5′-PTMs can be designed to repair mutations in the β-globin transcript involved in sickle cell anemia and β-thalassemia while providing insight into considerations for competition betweentrans- versuscis-splicing in mammalian cells. Target transcripts with impairedcis-splicing capabilities, like those produced in some forms of β-thalassemia, are more efficiently repaired viatrans-splicing than targets in whichcis-splicing is unaffected as with sickle β-globin. This study reveals desirable characteristics in substrate RNAs fortrans-splicing therapeutics as well as provides an opportunity for further exploration into general splicing mechanisms via 5′-PTMs.