High throughput screen for regulators of globin gene ex*

描述（由申请人提供）：镰状细胞病（SCD）是由β珠蛋白基因中的单个氨基酸取代ValSGIn引起的常见疾病。由于过去60年来的巨大科学进步，我们现在对SCD的分子病理生理学有了非常详细的了解。不幸的是，新疗法的开发进展缓慢。有效治疗的前景似乎很有希望，因为广泛的证据表明，胎儿血红蛋白的重新激活可以防止缺氧血红蛋白的聚合，这是SCD的中心异常。在过去，在多孔板的每个孔中高通量筛选多种转录物是不可行的。我们已经开发了一种检测7个不同的珠蛋白转录本和GAPDH的表达，在每个孔的多孔板具有高保真度和低成本，使我们能够筛选在高通量的化合物，差异调节珠蛋白基因的表达的小分子库。我们设计了我们的检测方法来分析珠蛋白基因在体外培养的原代人红系祖细胞中的表达。将RNA捕获在oligo-dT包被的384孔板（RNAture）上并逆转录。通过连接介导的PCR扩增每个感兴趣基因的cDNA，将标记的扩增子捕获在荧光微球（Luminex）上，并通过高速流式细胞术检测不同扩增子的相对丰度。RNA纯化、连接介导的扩增、荧光珠检测和数据采集的整个过程都可以自动化。到目前为止，我们已经证明，我们可以测量所有七个珠蛋白转录的表达在多孔格式，我们可以检测相对诱导γ珠蛋白表达的胎儿血红蛋白的已知诱导剂。在该提案中，我们描述了该测定的优化、验证和自动化以及试点小分子筛选的性能。与哈佛和麻省理工学院布罗德研究所的Stuart Schreiber博士的实验室合作，我们建议筛选富含FDA批准的和其他已知生物活性化合物的小分子库和整理的HDAC抑制剂库。完成这些研究后，我们希望能够鉴定出少量激活胎儿血红蛋白的化合物，以供进一步研究。此外，我们希望在中试筛选中验证该测定法，为更广泛的小分子文库筛选做准备。

DESCRIPTION (provided by applicant): Sickle cell disease (SCD) is a common disease caused by a single amino acid substitution, ValSGIn, in the beta globin gene. Due to tremendous scientific advances over the past 60 years, we now understand the molecular pathophysiology of SCD in exquisite detail. Unfortunately, progress in the development of novel therapies has been slower. Prospects for an effective therapy appear promising as extensive evidence demonstrates that reactivation of fetal hemoglobin prevents polymerization of deoxygenated hemoglobin, the central abnormality in SCD. In the past, a high throughput screen for multiple transcripts in each well of a multiwell plate has not been feasible. We have developed an assay that detects seven distinct globin transcripts and GAPDH expression in each well of a multiwell plate with high fidelity and at low cost, enabling us to screen libraries of small molecules in high throughput for compounds that differentially regulate expression of the globin genes. We have designed our assay to analyze expression of globin genes in primary human erythroid progenitor cells cultured in vitro. RNA is captured on oligo-dT coated 384-well plates (RNAture) and reverse transcribed. The cDNA for each gene of interest is amplified by ligation mediated PCR, labeled amplicons are captured on fluorescent microspheres (Luminex), and the relative abundance of different amplicons is detected by high speed flow cytometry. The entire process of RNA purification, ligation mediated amplification, fluorescent bead detection, and data acquisition is amenable to automation. To date, we have demonstrated that we can measure expression of all seven globin transcripts in multiwell format, and we can detect the relative induction of gamma globin expression by known inducers of fetal hemoglobin. In this proposal, we describe the optimization, validation, and automation of this assay as well as the performance of a pilot small molecule screen. In collaboration with the laboratory of Dr. Stuart Schreiber at the Broad Institute of Harvard and MIT, we propose to screen a library of small molecules enriched in FDA approved and other known bioactive compounds and a library of collated HDAC inhibitors. Upon completion of these studies, we hope to have identified a small number of compounds that activate fetal hemoglobin for further investigation. Furthermore, we hope to have validated the assay in a pilot screen in preparation for more extensive screens of small molecule libraries.