High throughput screen for regulators of globin

描述(申请人提供)：镰状细胞病(SCD)是一种常见的疾病，由β珠蛋白基因中的单一氨基酸替换ValSGIn引起。在过去的60年里，由于巨大的科学进步，我们现在对SCD的分子病理生理学有了细致的了解。不幸的是，新疗法的开发进展缓慢。随着广泛的证据表明，胎儿血红蛋白的重新激活可以防止脱氧血红蛋白的聚合，这是SCD的中枢异常，因此有效治疗的前景似乎很有希望。在过去，对多孔板的每个孔中的多个转录本进行高通量筛选是不可行的。我们已经开发出一种方法，可以高保真、低成本地在多孔板的每一孔中检测七个不同的珠蛋白转录本和GAPDH表达，使我们能够高通量筛选小分子文库，寻找差异调节珠蛋白基因表达的化合物。我们设计了一种分析体外培养的原代人红系祖细胞珠蛋白基因表达的方法。RNA被捕获在寡聚脱氧核糖核酸涂层的384孔板(RNAture)上，并被逆转录。通过连接介导的聚合酶链式反应扩增每个目的基因的cDNA，在荧光微球(Luminex)上捕获标记的扩增片段，并用高速流式细胞仪检测不同扩增片段的相对丰度。整个RNA提纯、连接介导的扩增、荧光珠检测和数据采集的整个过程都是自动化的。到目前为止，我们已经证明了我们可以以多孔形式测量所有七个珠蛋白转录本的表达，并且我们可以检测已知的胎儿血红蛋白诱导剂对伽马珠蛋白表达的相对诱导。在这项提案中，我们描述了这种分析的优化、验证和自动化，以及中试小分子筛查的性能。与哈佛大学和麻省理工学院博德研究所的斯图尔特·施赖伯博士的实验室合作，我们建议筛选一个富含FDA批准的和其他已知生物活性化合物的小分子文库和一个整理的HDAC抑制剂文库。在完成这些研究后，我们希望能鉴定出少量能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分析，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.