Investigating post-transcriptional regulation of BCL11A to develop therapeutic approaches to ß-hemoglobinopathies – PREDA

Beta-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult hemoglobin beta-chain. The clinical severity is alleviated by the persistent production of the fetal beta-like gamma-globin chain in adulthood. BCL11A is the major fetal gamma-globin transcriptional repressor, thus modulation of the BCL11A-mediated fetal gamma-globin repression is a promising approach to increase cellular levels of gamma-globin. The mechanisms underlying developmental regulation of BCL11A remain currently elusive although it is proposed to involve post-transcriptional regulation steps. Alternative splicing of BCL11A mRNAs results in at least 4 transcripts that encode distinct protein isoforms (BCL11A-XS, BCL11A-S, BCL11A-L and BCL11A-XL). BCL11A-XL can silence gamma-globin expression, whereas BCL11A-L/S/XS lack this activity. Here, we hypothesize that manipulation of splicing could represent a means of unleashing the BCL11A transcriptional repressive activity on fetal gamma-globin. Our proposal aims to identify cis-splice regulatory sequences controlling BCL11A splicing to change the balance between protein isoforms and restore gamma-globin expression in adult erythroid cells. To this aim, we will first use third-generation sequencing to perform an exhaustive analysis of BCL11A transcript diversity in fetal vs adult erythroid cells. Then, we will reprogram BCL11A splicing using antisense oligonucleotides (ASOs) targeting BCL11A splice regulatory sequences to decrease the production of the BCL11A-XL isoform and activate fetal hemoglobin expression. The best-performing ASOs will be tested in cells obtained from beta-hemoglobinopathy patients to correct the pathological phenotype. Overall, this project will give insights in BCL11A splicing regulation and improve our understanding of BCL11A activity on globin gene expression. Furthermore, the use of ASOs to reprogram the splicing of BCL11A will allow the development of novel therapeutic strategies for beta-hemoglobinopathies.