PROJECT SUMMARY/ABSTRACT The goal of the proposed five-year training plan is the development of my independent research career as an academic adult hematology physician-scientist studying red cell biology and hemoglobin regulation. I have completed internal medicine residency and hematology/oncology fellowship training at the University of Pennsylvania, and I am currently a Senior Fellow/Program Scholar who will transition in July 2021 to an Instructor and attending physician in the Division of Hematology/Oncology at UPenn. I am specifically seeking to develop and refine the skills that will be required for a successful career as an independent investigator, including expertise in gene regulation, signaling pathways, functional genomics, and bioinformatics. My overarching goal is to improve therapeutic approaches for sickle cell disease (SCD) via study of key signaling pathways that regulate expression of the fetal form of hemoglobin. My mentor for this award is Dr. Gerd Blobel, an internationally recognized leader in erythroid gene regulation and hemoglobin switching. To add depth and breadth to my scientific and career guidance, I have assembled a Mentoring Committee composed of physician-scientists from diverse and complementary fields. I will have the full resources of UPenn and the Children’s Hospital of Philadelphia available for the completion of my research and career development goals. The goal of this proposal is to elucidate the molecular mechanisms of PP6C, a novel regulator of fetal hemoglobin, to improve upon current treatments for SCD and beta-thalassemia. SCD afflicts millions of people worldwide and can lead to severe complications including acute chest syndrome, stroke, avascular necrosis of bone, and nephropathy. Although increasing levels of fetal hemoglobin (HbF) significantly reduces cell sickling and SCD-related morbidity and mortality, effective HbF pharmacologic induction has been an elusive goal. To this end, I recently carried out a CRISPR-Cas9 based screen to identify additional potentially druggable molecules to increase HbF production; this screen uncovered the protein phosphatase PP6C as a novel HbF regulator. This proposal will explore PP6C-regulated pathways with both hypothesis-driven and unbiased approaches and will investigate suitability of PP6C as a target for HbF induction. These objectives will be achieved via three Specific Aims: to elucidate key mechanistic pathways in the regulation of HbF by PP6C, to explore potential cooperativities of PP6C with other HbF regulatory pathways utilizing CRISPR-Cas12a-based techniques; and to test the role of PP6C-mediated HbF regulation in SCD and in vivo models. The outcome of these studies will deepen our understanding of signaling pathways that govern HbF expression and unveil new therapeutic opportunities in SCD. Completion of these aims will consolidate my experience in models of hemoglobin switching, further my training in bioinformatics and functional genomics, ...