Dissecting the transcriptional and epigenetic regulation of HEMOglobin to unravel novel CURativE options for beta-hemoglobinopathies – HEMOCURE

Beta-hemoglobinopathies are caused by mutations affecting the production of the adult hemoglobin beta-chain. Persistence of fetal gamma-globin chain synthesis in adult life substantially ameliorates the clinical phenotype of beta-hemoglobinopathy patients.
Currently, allogenic hematopoietic stem cell transplantation is the only definitive therapy for patients affected by severe beta-hemoglobinopathies. Transplantation of autologous, genetically modified hematopoietic stem cells represents a therapeutic option for patients lacking a suitable donor. Gene therapy strategies currently in clinics include the transplantation of hematopoietic stem cells transduced with an integrating lentiviral vector expressing a functional beta-globin gene. Genome editing approaches based on the use of site-specific nucleases have been explored by many groups, including ours. Clinical trials have been recently initiated exploiting genome editing to down-regulate BCL11A, a master repressor of fetal hemoglobin, via the inactivation of its erythroid-specific enhancer. However, a non-trivial genotoxicity risk associated with the use of these approaches raises concerns when applied in the clinics.
In this project, we will use cutting-edge epigenome editing technologies to dissect the molecular mechanisms underlying gamma- and beta-globin gene regulation, and to develop novel potential therapeutics for beta-hemoglobinopathies. We will identify critical regions in two regulatory elements that control gamma-to-beta globin switch during development, such as the BCL11A erythroid-specific enhancers and the gamma-globin gene promoters. We will identify epigenetic marks that are differentially deposited in fetal or adult cellular models at these regions to secure silencing of gamma-globin and activation of beta-globin. We will then both use previously established designer epigenome modifiers (DEM) and develop novel effectors to alter these epigenetic marks in order to restore gamma-globin expression in a cellular model of adult erythropoiesis. To this end, we will use the DEM technology to erase activating marks and deposit repressive epigenetic marks at the BCL11A enhancers in order to inactivate these elements. We will then pursue a similar approach to deposit activating epigenetic marks at the gamma-globin promoters. We anticipate an increase in gamma-globin expression as a result of both the inactivation of the BCL11A enhancers and the direct activation of its own promoter. The best-performing reagents selected from the in vitro model will be tested in primary human cells from healthy donors to identify the most efficient to induce fetal globin expression in the context of clinically-relevant cells. Ultimately, the most efficient reagents will be used to reactivate fetal globin in patient-derived cells and their safety profile will be thoroughly evaluated to reveal the potential of this approach for clinical translation.
Overall, the knowledge acquired in this project will be instrumental to develop novel therapeutic approaches aimed at restoring gamma-globin expression in patients affected by beta-hemoglobinopathies.