PNAs targeting BCL11A at the miR-210 binding sites down-regulate BCL11A and up-regulate gamma-globin mRNA

In β-thalassemia reactivation of γ-globin genes and production of fetal hemoglobin (HbF) is clinically relevant for compensating for the loss of β-globin chain. Interestingly, γ-globin gene expression is under a strong negative control performed by the transcription repressors BCL11A, KLF-1, MYB, Oct-1 and LYAR. Among these, BCL11A plays a pivotal role and it was recently shown by genome-wide association studies as the major repressor of HbF expression. MicroRNAs (miRNAs, miRs) are a category of conserved, short RNAs that target specific mRNA-sequences, leading to a post-transcriptional negative control. We found that a coding sequence of BCL11A mRNA contains a miR-210 binding site, located at the level of a coding BCL11A mRNA sequence. Forced expression of miR-210 leads to decrease of BCL11A mRNA and increase of γ-globin mRNA content in erythroid cells, including erythroid precursors isolated from γ-thalassemia patients. Our results suggest that the coding mRNA sequence of BCL11A can be targeted by miR-210. Accordingly, as a novel strategy to increase γ-globin gene expression, in this study we employed Peptide Nucleic Acids (PNAs) to inhibit BCL11A. We have targeted the miR-210 BCL11A binding region with PNAs mimicking miR-210 sequence and PNAs mimicking the sequence perfectly pairing the BCL11A-miR-210 binding site. The results obtained demonstrate that treatment of erythroid cells with these PNAs leads to a decrease of BCL11A and an increase of γ-globin gene expression. This field of research is of top interest, since several approaches are focused on pharmacologic inhibition of the expression of γ-globin gene repressors, leading to increased γ-globin gene expression.