Molecular Basis of Diamond-Blackfan anemia

描述（由申请人提供）： 本申请的长期目标是：i）更好地了解Diamond-Blackfan贫血（DBA）背后的基本分子病理学，以及ii）开发DBA的新治疗模式。第一个目标将使用核糖体蛋白（RP）S19、其信使RNA（mRNA）及其基因来实现。在25%的DBA患者中发现RPS 19突变，但RPS 19的作用机制仍不清楚。具体而言，该项目旨在鉴定与RPS 19和/或其mRNA相互作用的因子。将分析体外转录的RPS 19 mRNA在红系和髓系细胞系中的相互作用伴侣。系统还将用于在UT-7和K562细胞中化学交联后鉴定和分离特异性RPS 19相互作用蛋白或RNA。建立的系统也将用于研究突变型RPS 19对红系细胞系中剪接的影响。与RPS 19或其mRNA相互作用的因子可以阐明突变型RPS 19介导DBA和可能的其他骨髓衰竭综合征的途径。这些新发现的途径也可以作为未来治疗干预的靶点。 对于新的治疗方式，将对具有破坏的Rps 19基因的DBA小鼠模型进行基因转移。分离来自Rps 19 +/-小鼠的红系前体细胞（Lin-，c-kit+）并用Rps 19-GFP慢病毒构建体转导。重新引入表达Rps 19的细胞，并在体内监测对红系细胞产生、Rps 19表达和小鼠的一般健康/生长的影响。 该项目将利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.