Regulation and Function of a Human Embryonic Globin

描述（由申请人提供）：该提案继续了一个高效的项目，致力于开发利用内源性或外源性基因编码人类胚胎珠蛋白的地中海贫血和镰状细胞病的新治疗策略。关键分析已经确定，含有-珠蛋白亚基的异四聚体血红蛋白具有生理上适当的o2结合特性，并且出乎意料地显示出高度显著的抗镰状红细胞凝集活性。其他研究表明，转录后机制通过影响-珠蛋白编码mRNA的功能特性，在调节-珠蛋白表达中发挥重要作用。这项重要的工作扩展到三个新的特定目标，研究胚胎的天然结构如何？-珠蛋白mRNA在成年期确定的红细胞中表达时决定其稳定性和翻译。Aims利用了利用申请人实验室经验的技术，以及已建立的方法，这些方法已被修改以满足本提案的实验要求。目的：我将定义特定的顺式作用元素，这些元素决定了？-球蛋白mRNA，通过一种新的基于细胞培养的饱和诱变策略。序列替换优化这些元素的功能——产生超稳定？-珠蛋白mrna -随后将使用创新的体内SELEX方法进行鉴定。Aim II将通过抑制40S核糖体扫描或80S核糖体进程，确定-珠蛋白5'UTR和编码区内的一级和二级结构基序，从而抑制其在终代红细胞中的翻译。然后，这些元素将以一种使效率最大化的方式进行改变。-珠蛋白是由其同源mRNA在体内完整细胞中翻译而来的。第三部分将测试两种独立的增强方法。通过结合在Aims I和II中鉴定的位点特异性功能增强序列替换，以及通过工程增加有效？球蛋白翻译。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发育表达中的关键作用，(b)产生一个？-球蛋白mRNA，经过转录后优化，可产生具有治疗效用的球蛋白蛋白，最重要的是，(c)描述了一种高度创新的方法，可用于增强其他球蛋白和非球蛋白基因的表达。拟议的研究是对转录后过程在调节基因表达中的作用的不断发展的理解的关键一步，以及它们可以被操纵以治疗地中海贫血和镰状细胞性贫血患者的方式。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——包括那些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分子策略的新治疗范式。目前的建议描述了一种新的方法，其中调节珠蛋白mrna的稳定性和翻译效率的过程被操纵以获得治疗优势。这种策略特别有吸引力，因为它可以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.