RNA repair restores hemoglobin expression in IVS2–654 thalassemic mice

Abstract

Repair of ␤-globin pre-mRNA rendered defective by a thalassemiacausing splicing mutation, IVS2-654, in intron 2 of the human ␤-globin gene was accomplished in vivo in a mouse model of IVS2-654 thalassemia. This was effected by a systemically delivered splice-switching oligonucleotide (SSO), a morpholino oligomer conjugated to an arginine-rich peptide. The SSO blocked the aberrant splice site in the targeted pre-mRNA and forced the splicing machinery to reselect existing correct splice sites. Repaired ␤-globin mRNA restored significant amounts of hemoglobin in the peripheral blood of the IVS2-654 mouse, improving the number and quality of erythroid cells.oligonucleotides ͉ RNA splicing ͉ thalassemia ͉ therapy ͉ morpholino oligomers O ne of the most common inherited diseases, ␤-thalassemia, is caused by defects in the ␤-globin gene that affect production of ␤-globin, a subunit of hemoglobin (1). Current therapy consists of frequent blood transfusions combined with iron chelation (2). The only cure, bone marrow transplantation, is limited by the scarcity of suitable histocompatible donors (3). Although more than 200 mutations cause the disease, some of the most common ones induce aberrant splicing of ␤-globin pre-mRNA, interfering with proper translation of ␤-globin protein. The IVS2-654 (C Ͼ T) mutation creates an aberrant 5Ј splice site and activates a cryptic 3Ј splice site within intron 2 of the ␤-globin pre-mRNA, leading to retention of the intron fragment in the spliced mRNA even though the correct splice sites remain undamaged and potentially functional ( Fig. 1A) (4). The retained fragment prevents proper translation of ␤-globin, leading to hemoglobin deficiency and ␤-thalassemia. The IVS2-654 mutation is a common cause of thalassemia in Thailand and other countries of Southeast Asia (5). Work in this laboratory showed that splice-switching oligonucleotides (SSOs), which block aberrant splice sites in IVS2-654 and other pre-mRNAs (IVS1-5, IVS1-6, IVS1-110, IVS2-705, and IVS2-745) as well as in the coding sequence (HbE) of the ␤-globin gene, force the splicing machinery to reselect the existing correct splice sites, repairing the splicing pattern of ␤-globin pre-mRNA. This repair, which restores production of correctly spliced ␤-globin mRNA and protein, was accomplished in several in vitro systems and ex vivo in erythroid progenitor cells from thalassemic patients (6)(7)(8)(9)(10)(11)(12). In this study, we investigated the effectiveness in thalassemic splicing correction of a modified morpholino oligomer, SSO 654-P005, in a mouse model of IVS2-654 ␤-thalassemia. Results and DiscussionTo be effective in splicing repair, SSOs must hybridize tightly to the targeted splicing elements, such as aberrant splice sites, and prevent their recognition by the splicing factors. Furthermore, the resulting double-stranded structures must not be recognized by RNase H, which degrades RNA in RNA-DNA duplexes (13). These conditions are satisfied in cell culture and in vivo by modified oligomers with various backbones, includi...

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