The aim of this proposal is to develop novel, tailored agents than can induce the generation of human fetal red blood cells from adult hematopoietic stem cells by reprogramming ?-type globin gene regulation. Such agents can be potentially applied to the treatment of ?-globin disorders: sickle cell disease (SCD) and ?- thalassemia. Since fetal 3-globin chains inhibit red cell sickling in SCD, therapeutic agents that increase 3-globin production are predicted to ameliorate both the symptoms and pathophysiology associated with the disease. Recent efforts in drug therapy for ?-thalassemia have also focused on stimulation of 3-globin gene expression, but with only limited success. Because the effects of 3-globin inducers such as hydroxyurea, 5- azacytidine, and butyrates are mediated either through non-specific cytotoxicity or global changes in epigenetic chromatin modification, those agents would cause a variety of unfavorable cellular and systemic side effects. Those include non-selective global gene de-repression, death of rapidly proliferating cells (causing bone marrow suppression), carcinogenicity, and teratogenicity. Those adverse effects would limit therapeutic dosages, application, and efficacy of those agents. Therefore, more effective and safer 3-globin inducing agents are needed. This proposal is focused on developing novel classes of therapeutic agents that inhibit a specific repressor of human 3-globin gene expression. TR2 and TR4, orphan nuclear receptors without any known ligand, have been recently identified as embryonic ?- and fetal 3-globin gene repressors, and would be outstanding targets for molecular intervention therapy for ?-globin disorders. In this proposal, two different strategies will be adopted to modulate the activity of TR2 and TR4. The first will be to develop highly efficient small interfering RNA (siRNA) duplexes that specifically knock-down TR2, TR4, or their potential co-regulatory proteins by RNA interference. The second strategy will be to identify low-molecular-weight chemical ligands, either synthetic or natural, that can inhibit the repressor activity of TR2 and TR4, or even convert them from repressors to activators as is observed with many other nuclear receptors upon ligand binding. To detect ligands, a rapid, sensitive cell-based assay will be developed and then used for high-throughput screening of a large-scale random synthetic compound library, as well as for screening of various mouse tissue extracts to search for natural ligands, which will be then affinity-purified and identified by mass spectrometry. Once such anti- TR2/TR4 agents, either siRNAs or chemical ligands, are developed, they could immediately serve as either direct or lead therapeutic agents for ?-globin disorders. Both primary mouse and human erythroid cells differentiated ex vivo from adult hematopoietic progenitors will be explored as possible model systems to test those agents for their ability to induce """"""""fetal"""""""" erythroid cells with high-level 3-globin expression as an initial step toward therapeutic application for the treatment of SCD and ?-thalassemia.
The goal of this proposal is to develop novel drugs that can be safely used to treat patients with sickle cell disease (SCD) or 2-thalassemia. SCD affects 70,000-90,000 people in the U.S., and millions worldwide, causing severe pain, organ damage, and premature death (patients commonly die in their 40s). Severe forms of 2-thalassemia affect 800-1,000 people in North America, and 60,000 newborns annually worldwide, causing severe anemia that requires life-long blood transfusion for survival. Currently, only a few drugs are available to treat SCD, and these only modestly ameliorate the symptoms in only about half of the patients, but there are no current treatments for severe forms of 2-thalassemia. The only therapeutic option to cure these devastating diseases is stem cell transplantation that is often hampered by risk of therapy-related death, high cost, and lack of donor availability. We propose to develop a drug that acts on a specific protein that our work previously showed inhibits fetal hemoglobin production.
