Identification of Novel Regulators of Fetal Hemoglobin Expression

Harvard Medical School, Boston MA

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Abstract

? DESCRIPTION (provided by applicant): Normal human development involves a switch in expression from fetal-to-adult (?-to-ß) hemoglobin during the postnatal period. The major ß-globin disorders, sickle cell disease and ß-thalassemia, are significant sources of morbidity and mortality and among the most common genetic diseases in the world. Clinical observation that the morbidity associated with ß-globin disorders is ameliorated by elevated levels of fetal hemoglobin (HbF) has provided the rationale for reactivation of the silenced HbF in adult life for therapeutic benefit. Genome-wide association studies (GWAS) and subsequent functional and genetic approaches identified BCL11A as a bona fide repressor of HbF. While BCL11A is dispensable for red cell production, it is required for normal mouse development (and B-cell production), which may present a challenge for inhibiting BCL11A with systemically administered drugs (i.e. small molecules). Further, BCL11A is a transcription factor, a class considered undruggable. This project aims to identify novel genes in BCL11A-dependent and BCL11A-independent pathways that regulate HbF to further elucidate the mechanisms of human globin switching and potentially uncover targets more amenable to drug development. A genome-wide shRNA screen performed in this laboratory with primary human erythroid progenitor cultures derived from CD34+ progenitor cells has provided additional candidate genes involved in HbF regulation. The primary screen implicated RIOK3, an erythroid-specific kinase, as a negative regulator of HbF, which may lie in a BCL11A-dependent or BCL11A-independent pathway. I will further characterize the role of RIOK3 in HbF regulation through shRNA and clustered regularly interspaced short palindromic repeats (CRISPR) knockdown/knockout with phenotypic rescue in CD34+ cells. Preliminary data also implicated PRDM11 as an activator of ß-globin. I will further characterize the role of PRDM11 in globin switching and its synergistic effect with BCL11A by shRNA and CRISPR knockdown/knockout with phenotypic rescue in both murine erythroleukia (MEL) cells and primary human CD34+ cells in the presence/absence of BCL11A. Collectively, these studies will deepen our understanding of the fetal-to-adult switch and ?-globin gene regulation, which is critical for HbF reactivation as a therapeutic option.