Genome Editing approaches to reactivate gamma-globin for the Treatment of ß-Hemoglobinopathies – GETH

ß-hemoglobinopathies (Sickle cell disease and ß-thalassemias) are genetic anemias affecting thousands of newborns annually worldwide, and representing an increasing health problem in European and Asian countries, including France. ß hemoglobinopathies are caused by mutations affecting the adult hemoglobin expression and are currently treated by red blood cell (RBC) transfusion and iron chelation regiments. For patients affected by severe ß-hemoglobinopathies, allogenic hematopoietic stem cell (HSC) transplantation is the only definitive therapy. Transplantation of autologous, genetically corrected HSCs represents an alternative therapy for patients lacking a suitable bone marrow donor. The clinical course of ß-thalassemias and Sickle cell disease (SCD) is ameliorated in the presence of elevated expression of the fetal gamma-globin genes. Unfortunately, pharmacological treatments showed high levels of toxicity, whereas knock-down of nuclear factors critical for fetal globin silencing, such as BCL11A, which is essential for hematopoietic development, are likely to have pleiotropic effects. Naturally occurring large deletions encompassing ß- and delta-globin genes in the globin gene cluster and point mutations in the gammaA- and gammaG-globin promoters, defined as Hereditary Persistence of Fetal Hemoglobin (HPFH) traits, result in increased fetal hemoglobin (HbF) expression ameliorating both thalassemic and SCD clinical phenotypes. The goal of the proposed project is the development of novel therapeutic strategies based on nuclease-mediated genome-editing approaches finalized to re-activate fetal globin expression in the erythroid progeny of ß-thalassemic and SCD HSC. Preliminary mutational analyses of individuals carrying HPFH deletions and ß-thalassemic patients revealed the presence of putative HbF silencers in the ß-globin gene cluster, binding the fetal globin repressor BCL11A. In this project, sequence-specific nucleases will be used to specifically disrupt these cis-regulatory functional elements applying a “Chinese box” strategy focused to define the smallest genomic region responsible of HbF silencing. Additionally, we will introduce HPFH point mutations and binding motifs of gamma-globin activators in the gammaA- and gammaG-globin promoters to create de novo binding sites for nuclear factors promoting HbF expression. This approach will give insights into globin regulation and provide the molecular bases for the development of a novel therapeutic strategy for ß-globin disorders.