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，致力于利用编码人胚胎球蛋白的内源性或外源性基因开发地中海贫血和镰状细胞病的新治疗策略。关键的分析已经建立，异源四聚体血红蛋白纳入-珠蛋白亚基具有生理上适当的O2-结合特性，出乎意料的是，也显示出非常显着的抗镰状活性。进一步的研究表明，转录后机制在调节β-珠蛋白表达中发挥重要作用，通过影响其编码mRNA的功能特性。这项重要的工作是扩展在三个新的具体目标，调查如何胚胎的原生结构？珠蛋白mRNA决定了其在成体阶段定形红系细胞中表达时的稳定性和翻译。目标利用了利用申请实验室经验的技术，以及已修改的既定方法，以解决本提案的实验要求。目的我将定义特定的顺式作用元件，决定的稳定性？珠蛋白mRNA，通过一种新的细胞培养为基础的饱和诱变策略。优化这些元件功能的序列替换--产生超稳定的？珠蛋白mRNA--随后将使用创新的体内SELEX方法进行鉴定。目的II将确定初级和二级结构基序内的-珠蛋白5 '非翻译区和编码区，抑制其翻译在定形红细胞通过抑制40 S核糖体扫描或80 S核糖体合成能力。然后，这些元素将以最大化效率的方式进行改变，珠蛋白在体内完整细胞中由其同源mRNA翻译而来。目标III将测试两种独立的方法来增加？通过结合目的I和II中鉴定的位点特异性功能增强序列取代，并通过工程化增加有效？珠蛋白翻译。所有三个目标的结果将（a）详细说明转录后过程在调节人球蛋白发育表达中的关键作用，（B）产生一个？珠蛋白mRNA，其经转录后优化以产生具有经证实的治疗效用的珠蛋白，并且最重要的是，（c）描述了可用于增强其它珠蛋白和非珠蛋白基因的表达的高度创新的方法。拟议的研究包括一个关键的一步，在不断发展的理解的作用，转录后过程中发挥调节基因表达，以及如何在地中海贫血和镰状细胞贫血患者的治疗优势，他们可以被操纵的方式。公共卫生相关性：尽管最近在临床护理方面有所改善，但影响人类球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率的主要原因。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。目前的提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，因为它可以应用于增强现有疗法的有效性，包括基因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.