Sodium Butyrate and Valproic Acid as Splicing Restoring Agents in Erythroid Cells of beta-Thalassemic Patients

Abstract

Background: beta-Thalassemia is a common autosomal recessive disorder in human caused by a defect in beta-globin chain synthesis. The most common mutations causing beta-Thalassemia have been found to be splicing mutations. Most of which activate aberrant cryptic splicing/sites without complete disruption of normal splicing. IVSI-110 mutation, a common splicing mutation, leads to a 90% reduction of normal beta-globin synthesis and lead to blood transfusion dependency in the homozygote forms. However, modulation of splicing can be achieved by activation or suppression of transacting factors such as SR (Serine, Arginine) amino acids and hnRNPs (Heterogeneous ribonucleoprotein particle) through drugs. Objectives: The aim of this study was to investigate the effects of NaBu, isoBu and VPA drugs on restoration of splicing of IVSI-110 beta-Thalassemia pre-mRNA in human. Materials and Methods: Primary erythroid cells derived from IVSI-110 beta-Thalassemia patients were cultured ex vivo and differentiated in the presence of 0.5 and 1 mM of Na-Butyrate (NaBu), 0.5 mM Isobutyramide (isoBu) and 100 mu M Valproic acid (VPA). RT-PCR analysis was used to evaluate the effect of the drugs in correction of normal splicing in beta-globin mRNAs. Results: Following treatment with NaBu, isoBu and VPA, the level of normal beta-globin mRNA in Primary erythroid cells derived from IVSI-110 beta-Thalassemia patients, increased 1.7, 1.5, 1.4 fold, respectively relative to normal beta-globin mRNAs. Higher splicing restoration was achieved by NaBu, a histone deacetylase inhibitor, known to upregulate the expression of splicing factors. Conclusion: The results highlighted the therapeutic potential of splicing modulation for genetic diseases caused by splicing mutations.