PROJECT SUMMARY Sickle cell disease (SCD) is one of the most commonly inherited blood disorders world-wide with an annual cost of over 1 billion dollars in the United States alone. Currently, there is only one FDA-approved drug for managing SCD, hydroxyurea (HU). HU efficacy is attributed largely to augmented expression of gamma-globin in erythroid progenitors leading to increased production of fetal hemoglobin to ameliorate many complications of SCD. However, the increase in fetal hemoglobin may not be sufficient for some patients, and they may not benefit this therapy for reasons that are unknown. With limited alternative therapies available there is a need to improve efficacy of HU and develop new drugs for these patients. This project will provide new information about the mechanisms of HU efficacy that can offer pharmacogenetic targets to help personalize HU therapy and offer new therapeutic targets of fetal hemoglobin induction for developing additional treatments for SCD. Its novel concepts combined with a detailed training plan and mentorship from a highly accomplished team of translational and clinical researchers will also facilitate the career development of the principle investigator. Our previous work investigating modulators of HU pharmacology has identified that the cell membrane transporters urea transporter B (UTB) and organic cation/carnitine transporter1 (OCTN1) regulate the intracellular accumulation of HU. These transporters govern how much drug reaches intracellular targets and are associated with augmented fetal hemoglobin levels. We also found that intracellular miRNAs, miR- 148a, - 151-3p, and -494 are associated with HU therapy in erythroid cells of sickle cell patients in association with increased fetal hemoglobin level. Thus, we have identified two potential mechanisms through which HU may elicit an increase in fetal hemoglobin levels. To date, model limitations have hindered our studies to investigate these modulators in an in vivo model of SCD. Given these previous findings, we hypothesize that induction of fetal hemoglobin by hydroxyurea is modulated by transporters and miRNAs. In the rich research environment at University of Pittsburgh, using an in vitro model of erythropoiesis, we will test our hypothesis in the following two aims: 1) to determine whether UTB and OCTN1 transporters control uptake and efficacy of HU to induce fetal hemoglobin and 2) to determine whether miR-148a, -151-3p, and -494 control the efficacy HU to induce fetal hemoglobin. In a third aim, we will develop a novel in vivo model to dissect these and other mechanisms of hydroxyurea-mediated fetal hemoglobin in SCD. While conducting the translational research, the candidate will also take classes, participate in career development programs, learn technical skills in viral gene transduction and improve professional competencies related to communication, mentoring, teaching, management and leadership. Throughout the project mentoring by a team of senior-level hematology and stem cell researchers will help ensure that the candidate launches an independent research career and realize the long-term goal of improving treatment for SCD.

PROJECT NARRATIVE Hydroxyurea is the only FDA-approved drug for the treatment of sickle cell disease. Its efficacy is attributed to induction of fetal hemoglobin in red blood cells, which ameliorates disease complications. However, diminished efficacy of hydroxyurea in some patients limits its utilization and highlights the need for additional therapies. This application will investigate mechanisms of fetal hemoglobin induction by hydroxyurea in order to improve current therapy and identify molecular targets for developing new therapies for sickle cell disease. !