Reactivation of Fetal Gamma-globin Genes for the Treatment of Beta-globin Disorde

描述（由申请人提供）：本提案的目的是开发新的定制试剂，其可以通过重编程β型珠蛋白基因调控诱导成人造血干细胞生成人胎儿红细胞。这种药物可以潜在地应用于治疗<$-珠蛋白疾病：镰状细胞病（SCD）和<$-地中海贫血。由于胎儿β-珠蛋白链抑制SCD中的红细胞镰状化，因此预测增加β-珠蛋白产生的治疗剂可改善与该疾病相关的症状和病理生理学。最近在药物治疗地中海贫血方面的努力也集中在刺激3-珠蛋白基因表达上，但只有有限的成功。 由于3-珠蛋白诱导剂如羟基脲、5-氮杂胞苷和丁酸盐的作用是通过非特异性细胞毒性或表观遗传染色质修饰的整体变化介导的，因此这些试剂会引起各种不利的细胞和全身副作用。这些包括非选择性全局基因去抑制、快速增殖细胞死亡（导致骨髓抑制）、致癌性和致畸性。这些副作用将限制这些药剂的治疗剂量、应用和功效。因此，需要更有效和更安全的3-珠蛋白诱导剂。该提案集中于开发新型的治疗剂，其抑制人3-珠蛋白基因表达的特异性阻遏物。TR 2和TR 4是一种没有任何已知配体的孤儿核受体，最近被鉴定为胚胎和胎儿3-珠蛋白基因阻遏物，并且将是用于<$-珠蛋白疾病的分子干预治疗的突出靶点。在本研究中，将采用两种不同的策略来调节TR 2和TR 4的活性。第一个将是开发高效的小干扰RNA（siRNA）双链体，通过RNA干扰特异性地敲低TR 2，TR 4或其潜在的共调节蛋白。第二个策略是鉴定低分子量的化学配体，无论是合成的还是天然的，其可以抑制TR 2和TR 4的阻遏物活性，或者甚至将它们从阻遏物转化为激活物，如在配体结合时观察到的许多其他核受体。为了检测配体，将开发一种快速、灵敏的基于细胞的测定，然后用于大规模随机合成化合物文库的高通量筛选，以及用于筛选各种小鼠组织提取物以寻找天然配体，然后将其亲和纯化并通过质谱法鉴定。一旦开发出这种抗TR 2/TR 4试剂，无论是siRNA还是化学配体，它们都可以立即用作β-珠蛋白疾病的直接或主要治疗剂。将探索从成人造血祖细胞离体分化的原代小鼠和人红系细胞作为可能的模型系统，以测试这些试剂诱导具有高水平β-珠蛋白表达的“胎儿”红系细胞的能力，作为治疗SCD和β-地中海贫血的治疗应用的初始步骤。 公共卫生相关性：该提案的目标是开发可安全用于治疗镰状细胞病（SCD）或2-地中海贫血患者的新药。SCD在美国影响7万至9万人，全世界数百万人，造成严重疼痛，器官损伤和过早死亡（患者通常在40多岁时死亡）。严重形式的2-地中海贫血影响北美800- 1，000人，全世界每年有60，000名新生儿，导致严重贫血，需要终生输血才能生存。目前，只有少数药物可用于治疗SCD，并且这些药物仅在大约一半的患者中适度改善症状，但是目前没有针对严重形式的2-地中海贫血的治疗方法。治愈这些毁灭性疾病的唯一治疗选择是干细胞移植，这通常受到与治疗相关的死亡风险，高成本和缺乏供体可用性的阻碍。我们建议开发一种药物，作用于一种特定的蛋白质，我们以前的工作表明这种蛋白质会抑制胎儿血红蛋白的产生。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.