Project Summary/Abstract Reactivation of fetal hemoglobin expression in adult erythroid cells is a validated approach to genetic therapy of both b-thalassemia and sickle cell disease (SCD). As currently practiced, however, genetic therapy (either lentiviral delivery or CRISPR/Cas9 editing) cannot meet the large disease burden due to its reliance on myeloablative preconditioning of patients for hematopoietic reconstitution and the medically intensive nature of the procedure. Drug (small molecule) therapeutics are needed to treat the many patients with these disorders. The vision of our research is to use small molecule therapeutics to reactivate y-globin gene expression, and do so both robustly and safely. The most potent, validated repressor of g-globin expression is BCL11A. In this project we will extend our recent studies in which we leveraged new chemical methods of targeted protein degradation (TPD) to deplete BCL11A and reactivate HbF production. The project consists of two broad aims. First, we will determine the dynamics of globin gene transcription using a platform in which erythroid cells at different phases of the cell cycle can be isolated for nascent transcription analyses. Using acute TPD of BCL11A, we will then ascertain at which stage(s) of the cell cycle g- (HBG) globin is reactivated upon loss of the repressor. These data will provide temporal resolution of globin gene transcription at unprecedented resolution. In a second aim, we will explore the use of erythroid-specific E3 ubiquitin ligases, TRIM10 and TRIM58, to target degradation of BCL11A. The TRIM proteins will be directed to BCL11A with BCL11A-specific nanobodies. We will initiate discovery of ligands for the TRIM proteins in order to develop tool compounds for cell-specific TPD of BCL11A as a new therapeutic strategy. Our work will lay the groundwork for novel small molecule approaches for robust and safe reactivation of HbF for the hemoglobin disorders.