Molecular Basis of Diamond-Blackfan anemia

描述(由申请人提供)： 这项应用的长期目标是：i)更好地了解钻石-布莱克凡贫血(DBA)背后的基本分子病理学，以及ii)开发一种治疗DBA的新方法。第一个目标将通过使用核糖体蛋白(RP)S19及其信使RNA(MRNA)和ITS基因来实现。在25%的DBA患者中发现RPS19突变，但RPS19的作用机制尚不清楚。具体地说，该项目旨在确定与RPS19和/或其mRNA相互作用的因素。体外转录的RPS19mRNA将用于分析其在红系和髓系细胞系中的相互作用伙伴。该系统还将用于识别和分离UT-7和K562细胞中化学交联后特定的RPS19相互作用蛋白或RNA。一个已建立的系统也将用于研究突变的RPS19对红系细胞系剪接的影响。与RPS19或其mRNA相互作用的因素可能阐明突变的RPS19介导DBA和可能的其他骨髓衰竭综合征的途径。这些新发现的通路也可能成为未来治疗干预的靶点。对于新的治疗方式，带有中断的Rps19基因的DBA小鼠模型将接受基因转移。从Rps19+/-小鼠分离红系前体细胞(LIN-，c-kit+)，并用Rps19-GFP慢病毒载体转导。将表达Rps19的细胞重新引入，并将在体内监测对红系细胞产生、Rps19表达和小鼠一般健康/生长的影响。该项目将使用理想的环境和专业知识来实现这些目标。预期的结果将有助于阐明调控红细胞生成的机制，以及改善DBA患者的预后。

DESCRIPTION (provided by applicant): The long term objectives of this application are; i) to better understand the basic molecular pathology behind Diamond-Blackfan anemia (DBA), and ii) to develop a novel treatment modality for DBA. The first objective will be achieved using ribosomal protein (RP) S19, its messenger RNA (mRNA) and, its gene. RPS19 is found mutant in 25% of patients with DBA but the mechanisms by which RPS19 acts remain unknown. Specifically, the project aim at the identification of factors interacting with RPS19 and/or its mRNA. In vitro transcribed RPS19 mRNA will be analyzed for its interacting partners in erythroid and myeloid cell lines. A system will also be used to identify and isolate specific RPS19 interacting proteins or RNAs after chemical cross linking in UT-7 and K562 cells. An established system will also be used to study the effect of mutant RPS19 on splicing in erythroid cell lines. Factors interacting with RPS19 or its mRNA may clarify the pathway through which a mutant RPS19 mediates DBA and possibly other bone marrow failure syndromes. Such newly identified pathways may also serve as targets for future therapeutic intervention. For novel treatment modalities, a mouse model for DBA with a disrupted Rps19 gene will be subject to gene transfer. Erythroid precursor cells (Lin-, c-kit+) from the Rps19+/- mice are isolated and transduced with a Rps19-GFP lentiviral construct. Cells expressing Rps19 are re-introduced and the effect on erythroid cell production, Rps19 expression and general health/growth of mice will be monitored in vivo. The project will use an ideal environment and expertise to achieve these objectives. The expected results will help to shed light on mechanisms regulating erythropoiesis as well as to improve the outcome of patients with DBA.