Regulation and Function of a Human Embryonic Globin

描述(由申请人提供)：该提案延续了一项富有成效的计划，该计划致力于利用编码人类胚胎珠蛋白的内源性或外源性基因开发治疗地中海贫血和镰状细胞疾病的新策略。关键的分析已经证实，含有球蛋白亚基的异四聚体血红蛋白具有生理上合适的O2结合特性，并且出人意料地也显示出非常显著的抗病活性。更多的研究表明，转录后机制通过影响其编码的mRNA的功能特性，在调节珠蛋白的表达方面发挥了重要作用。这项关键的工作扩展到三个新的特定目标，研究胚胎-珠蛋白mRNA的天然结构如何决定其在成体阶段确定的红系细胞中表达时的稳定性和翻译。这些目标利用了利用申请实验室的经验的技术，以及已修改的既定方法，以解决这一提案的实验必要性。目的通过一种新的基于细胞培养的饱和诱变策略，定义决定β-珠蛋白mRNA稳定性的特定顺式作用元件。随后将使用一种创新的体内SELEX方法来确定优化这些元件功能的序列替换--产生超稳定的？-珠蛋白mRNAs。目的II将通过抑制40S核糖体扫描或80S核糖体加工来确定-珠蛋白5‘非编码区和编码区中的一级和二级结构基序，从而抑制其在确定的红系细胞中的翻译。然后，这些元件将以一种最大限度地提高在完整细胞中从其同源mRNA中翻译？-珠蛋白的效率的方式进行改变。AIM III将测试两种独立的方法，通过结合AIMS I和II中确定的特定部位的功能增强序列替代，以及通过提高有效的？-珠蛋白翻译所需的tRNAs的细胞水平，来增强原代确定的人类红系细胞中？-珠蛋白的表达。所有这三个目标的结果将(A)详细说明转录后过程在调节人类珠蛋白的发育表达中所起的关键作用，(B)产生经转录后优化的-珠蛋白mRNA，以产生具有治疗效用的珠蛋白蛋白，最重要的是，(C)描述一种可用于增强其他珠蛋白和非珠蛋白基因表达的高度创新的方法。这项拟议的研究是逐步了解转录后过程在调节基因表达中所起的作用，以及如何利用转录后过程在地中海贫血和镰状细胞性贫血患者中发挥治疗优势的关键一步。公共卫生相关性：尽管最近临床护理有所改善，但影响人类珠蛋白正常表达的先天性遗传疾病--包括导致镰状细胞病和地中海贫血的疾病--仍然是美国和国外发病率和过早死亡的主要原因。未来的治疗进展可能需要基于创新的分子策略的新的治疗范例。目前的提议描述了一种新的方法，在这种方法中，调节珠蛋白mRNAs稳定性和翻译效率的过程被操纵以获得治疗优势。这一策略特别有吸引力，因为它可以应用于增强现有疗法的有效性，包括基因再激活和基因替代方法。

DESCRIPTION (provided by applicant): This proposal continues a highly productive program that is committed to developing new therapeutic strategies for thalassemia and sickle cell disease utilizing endogenous or exogenous genes encoding human embryonic globins. Key analyses have established that heterotetrameric hemoglobins incorporating -globin subunits possess physiologically appropriate O2-binding properties and, unexpectedly, also display highly significant antisickling activity. Additional studies indicate the essential role that post-transcriptional mechanisms play in regulating -globin expression, through effects on the functional properties of its encoding mRNA. This critical work is extended in three new Specific Aims that investigate how the native structure of embryonic ?-globin mRNA dictates its stability and translation when expressed in adult-stage, definitive erythroid cells. The Aims utilize techniques that capitalize on the experience of the applicant laboratory, as well as established methods that have been modified to address the experimental imperatives of this proposal. Aim I will define specific cis-acting elements that dictate the stability of ?-globin mRNA, through a novel cell- culture based saturation mutagenesis strategy. Sequence substitutions that optimize the function of these elements--producing hyperstable ?-globin mRNAs--will subsequently be identified using an innovative in vivo SELEX approach. Aim II will identify primary and secondary structural motifs within the -globin 5'UTR and coding region that suppress its translation in definitive erythroid cells by inhibiting 40S ribosomal scanning or 80S ribosomal processivity. These elements will then be altered in a manner that maximizes the efficiency with which ?-globin protein is translated from its cognate mRNA in vivo in intact cells. Aim III will test two independent methods for augmenting ?-globin expression in primary definitive human erythroid cells, by combining the site-specific function-enhancing sequence substitutions identified in Aims I and II, and by engineering an increase in the cellular levels of tRNAs required for efficient ?-globin translation. The results from all three Aims will (a) detail the critical role that post-transcriptional processes play in regulating the developmental expression of human globins, (b) generate a ?-globin mRNA that is post-transcriptionally optimized to produce a globin protein with demonstrated therapeutic utility and, most importantly, (c) describe a highly innovative approach that can be used to augment the expression of other globin and nonglobin genes. The proposed research comprises a crucial step in an evolving understanding of the role that post- transcriptional processes play in regulating gene expression, and the manner in which they can be manipulated to therapeutic advantage in patients with thalassemia and sickle cell anemia. PUBLIC HEALTH RELEVANCE: Despite recent improvements in clinical care, congenital genetic disorders affecting the normal expression of human globins--including those responsible for sickle cell disease and thalassemia--remain a major cause of morbidity and premature mortality both in the United States and abroad. Future therapeutic advances are likely to require new treatment paradigms based upon innovative molecular strategies. The current proposal describes one new approach, in which processes that regulate the stability and translational efficiency of globin mRNAs are manipulated to therapeutic advantage. This strategy is particularly attractive because it can be applied to augment the effectiveness of existing therapies, including gene-reactivation and gene-replacement approaches.