Increased fetal hemoglobin (HbF) in cells of patients with b- hemoglobinopathies (b-thalassemia and sickle cell anemia) ameliorates the clinical symptoms of the underlying disease. Several pharmacologic agents have been used to stimulate HbF synthesis; e.g., treatment of patients with hydroxyurea resulted in higher HbF levels and a significant improvement in the clinical symptoms. Yet, there is a considerable interest in other, less toxic agents with even higher potential to increase HbF. So far only a handful of agents have been tested, mainly due to the lack of an appropriate experimental system that allows a rapid and accurate determination of the effect on relevant cells. We have developed a two-phase liquid culture system for growing erythroid progenitors derived from the peripheral blood of normal individuals and patients with hemoglobinopathies. We showed that the system recapitulates many aspects of erythropoiesis in vivo, including stimulation of HbF production by pharmacologic agents. The purpose of our research is to utilize this culture procedure and various biochemical, molecular and immunological methods to study the regulation of Hb production and for screening of agents for their HbF- stimulating potential with the final objective to produce an efficient, low toxicity therapy. The experiments include optimization of the conditions for stimulation of HbF production, followed by screening of various agents (including derivatives of hydroxyurea, aromatic fatty acids, hemoglobin and hemin) and subsequently studying their mode(s) of action. The latter studies will provide the rationale for further screening of additional agents and for their chemical modification in order to increase efficacy. Classification of the drugs according to functional and mechanistic considerations will enhance experiments with combinations of drugs belonging to different groups and, thus, expected to act synergistically. In addition, we are attempting to develop, based on the cell culture system, an assay for evaluating an individual patient's response to a particular drug or drug combination. We are also working with a newly developed robotic system for screening large numbers of compounds which may affect globin gene transcription; chemical libraries of thousands of compounds are now being screened by this method. We are testing compounds which score positively in this system in our culture system, as well as in rhesus primates (in conjunction with the NHLBI facility).
