Hemoglobin Switching: Genetics to Translation

主要的血红蛋白疾病、镰状细胞性贫血和B型地中海贫血给全球带来的健康负担是巨大的，而且预计还会增加。胎儿血红蛋白（HbF）的增加大大改善了这些疾病的发病率和死亡率。长期寻求的目标是在患有血红蛋白病症的成人中定向再激活HbF表达。通过对人类遗传学的研究， 了解控制胎儿（a2 Y2）到成人（a2 B2）转换的调节因子以及HbF沉默如何在成人中维持。所提出的工作的前提是，血红蛋白转换的基本发现提供了一个平台，用于建立基于机制的方法来识别小的 在成人红系细胞中诱导HbF的分子。如果在这一总体目标上取得成功，可能会开发新的疗法来治疗患有主要血红蛋白疾病的患者。这项建议包括三个独立但相互关联的目标。首先，编码BCL 11 A的基因，一个中心的 HbF表达的阻遏物，将使用高分辨率的新生转录作图，染色质占有率和putafive顺式调控元件的测定来探索。BCL 11 A表达或功能的下调提供了减轻HbF沉默的直接途径。这一目标的另一个目标是 还确定BCL 11 A基因中的遗传变异如何影响BCL 11 A自身的表达，因为这一知识可能导致直接下调BCL 11 A表达的方法。其次，将在培养的红系细胞中进行HbF的小分子诱导剂的高通量筛选， 将进一步评估有希望的“命中”，以确定受影响的途径和作为治疗剂的优化。 第三，将用全基因组短发夹RNA（shRNA）和开放阅读框（ORF）文库进行筛选，以鉴定用于在原代人CD 34衍生的红系祖细胞中诱导沉默HbF的新基因/途径。通过该提案中的多学科方法，将对HbF调节的基本发现转化为血红蛋白疾病患者的新疗法的识别和开发。

The global health burden ofthe major hemoglobin disorders, sickle cell anemia and B-thalassemia, is enormous and predicted to grow. Increased fetal hemoglobin (HbF) greatly ameliorates the morbidity and mortality of these disorders. A long sought goal is directed reactivation of HbF expression in adults with hemoglobin disorders. Largely through human genefic studies, substantial progress has been made in understanding the regulatory factors controlling the fetal (a2Y2) to adult (a2B2) switch and how HbF silencing is maintained in the adult. A premise of the proposed work is that fundamental findings on hemoglobin switching provide a platform for establishing mechanism-based approaches to the identification of small molecules that induce HbF in adult erythroid cells. If successful in this overall goal, new therapies may be developed for treatment of patients with the major hemoglobin disorders. This proposal encompasses three independent, but interrelated, aims. First, the mechanisms by which the gene encoding BCL11A, a central repressor of HbF expression, is regulated will be explored using high-resolution nascent transcription mapping, chromafin occupancy, and assays of putafive cis-regulatory elements. Down-regulafion of BCL11A expression or function provides a direct route to relief from HbF silencing. An addifional goal of this aim is also to determine how genefic variafion in the BCL1 IA gene influences expression of BCL11A itself, as this knowledge may lead to approaches to direct down-regulafion of BCL11A expression. Second, highthroughput screening for small molecule inducers of HbF will be performed in cultured erythroid cells, and promising "hits" will be evaluated further for the pathways affected and for optimization as therapeutics. Third, screens with genome-wide short hairpin RNA (shRNA) and open reading frame (ORF) libraries will be performed to identify novel genes/pathways for induction of silenced HbF in primary human CD34 derived erythroid progenitors. Through the mulfidisciplinary approaches in this proposal basic findings on HbF regulation will be translated to advance identificafion and development of new therapeutics for pafients with hemoglobin disorders.