Repair of a splicing defect in erythroid cells from patients with β-thalassemia/HbE disorder

Abstract

A HeLa cell line stably expressing the human β-globin gene carrying thalassemic mutations βE/IVS1-6 served as a thalassemia model for repair of aberrant splicing of ΒE/IVS1-globin pre-mRNA with antisense oligonucleotides. Treatment of βE/IVS1-6 HeLa cells with a morpholino oligonucleotide targeted immediately upstream of the aberrant 5′ splice site activated by the mutations resulted in an increase in the amount of correctly spliced βE-globin mRNA in a dose-dependent and sequence-specific fashion. The repaired βE-globin mRNA was stable and could be translated into full-length βE-globin polypeptide. Application of the same oligonucleotide to erythroid progenitor cells from two β-thalassemia/HbE patients resulted in an approximately 70% increase in correct βE-globin mRNA and 36% increase in hemoglobin E. The erythroid progenitor cells represent the actual targets for the clinical application of antisense repair of defective pre-mRNAs.