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

描述(申请人提供)：这项建议的目的是开发新的、量身定做的药物，通过重新编程β-型珠蛋白基因调控，从成人造血干细胞诱导产生人类胎儿红细胞。这类药物可能被应用于治疗伽玛珠蛋白紊乱：镰状细胞病(SCD)和β-地中海贫血。由于胎儿γ-珠蛋白链抑制了SCD患者的红细胞镰状，预计增加γ-珠蛋白产生的治疗药物将改善与该疾病相关的症状和病理生理学。最近在治疗β-地中海贫血的药物治疗方面的努力也集中在刺激伽马珠蛋白基因的表达上，但收效甚微。由于γ-珠蛋白诱导剂如羟基脲、5-氮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全局变化来介导的，这些诱导剂会引起各种不利的细胞和全身副作用。这些包括非选择性的全局基因抑制、快速增殖细胞的死亡(导致骨髓抑制)、致癌和致畸。这些副作用将限制这些药物的治疗剂量、应用和疗效。因此，需要更有效、更安全的丙种球蛋白诱导剂。这项提议的重点是开发新型治疗剂，抑制人类伽马珠蛋白基因表达的特定抑制物。TR2和TR4是孤儿核受体，没有任何已知的配体，最近被鉴定为胚胎和胎儿3-珠蛋白基因抑制因子，将成为分子干预治疗珠蛋白疾病的杰出靶点。在这项建议中，将采用两种不同的策略来调节TR2和TR4的活性。第一个是开发高效的小干扰RNA(SiRNA)双链，通过RNA干扰特异性地击倒TR2、TR4或它们潜在的协同调节蛋白。第二种策略将是确定低分子化学配体，无论是合成的还是天然的，可以抑制TR2和TR4的抑制活性，甚至可以将它们从抑制物转化为激活物，就像许多其他核受体与配体结合时观察到的那样。为了检测配体，将开发一种快速、灵敏的基于细胞的分析方法，然后用于大规模随机合成化合物文库的高通量筛选，以及各种小鼠组织提取物的筛选，以寻找天然配体，然后进行亲和纯化和质谱仪鉴定。一旦这种抗TR2/TR4的试剂被开发出来，无论是siRNAs还是化学配体，它们都可以立即成为治疗β-珠蛋白疾病的直接或主要治疗药物。将探索从成人造血祖细胞体外分化的原代小鼠和人类红系细胞作为可能的模型系统，以测试这些药物诱导具有高水平伽马珠蛋白表达的“胎儿”红系细胞的能力，作为治疗应用于治疗SCD和β-地中海贫血的第一步。

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 beta-type globin gene regulation. Such agents can be potentially applied to the treatment of gamma-globin disorders: sickle cell disease (SCD) and beta- thalassemia. Since fetal gamma-globin chains inhibit red cell sickling in SCD, therapeutic agents that increase gamma-globin production are predicted to ameliorate both the symptoms and pathophysiology associated with the disease. Recent efforts in drug therapy for beta-thalassemia have also focused on stimulation of gamma-globin gene expression, but with only limited success. Because the effects of gamma-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 gamma-globin inducing agents are needed. This proposal is focused on developing novel classes of therapeutic agents that inhibit a specific repressor of human gamma-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 beta-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 gamma-globin expression as an initial step toward therapeutic application for the treatment of SCD and beta-thalassemia.