Identification of Genes Involved in Beta- Thalassemia

描述（由申请人提供）：β-地中海贫血是一种单基因疾病，由β-珠蛋白基因突变引起，其特征为极其复杂的表型。人β-珠蛋白基因表达的减少干扰铁代谢并导致无效的红细胞生成、脾肿大和骨质减少。尽管发现了导致β-地中海贫血发展的遗传异常，但在这种疾病中受到干扰的细胞过程仍然没有解决。我们的工作假设是，β-珠蛋白表达的减少触发基因的表达水平的修改，有助于β-地中海贫血表型。因此，我们建议通过研究在正常和病理条件下其表达水平的改变来分离和表征在受这种疾病影响的组织中受到干扰的基因。最近，我们已经建立了第一个成年致死性库利氏贫血的小鼠模型，通过将小鼠β-珠蛋白基因（th 3/th 3）缺失的纯合子胎肝细胞移植到辐照野生型（wt）受体动物中。这些小鼠在移植后60天内死亡，原因是无效的红细胞生成和大量铁超负荷导致严重贫血。这种新型小鼠模型的开发使得首次鉴定β-地中海贫血病理生理学中涉及的关键基因成为可能。作为第一步，我们将使用微阵列分析来鉴定差异表达的基因，以比较（目的1）野生型动物、受中间型β地中海贫血和库利氏贫血影响的小鼠的脾、骨髓和肝脏来源的组织的基因表达谱。我们预测，通过微阵列分析分离的基因的过度表达或抑制将揭示这些基因和β-地中海贫血综合征之间的关键功能关系。这将在小鼠中进行测试，为了改变通过微阵列分析分离的单个基因的表达水平，我们将使用我们开发的抑制或过表达靶基因的慢病毒载体系统。我们将利用具有相同遗传背景的小鼠，从这些小鼠中分离出基因，以便一次修改一个参数（单个基因的表达水平）。因此，我们将利用一种新的慢病毒系统，以可控的方式，调节通过微阵列分析分离的内源性基因的表达。为了测试（目的2）它们的功能，在体内，在正常和病理条件下，胚胎干（ES）细胞将用该慢病毒系统转导，该慢病毒系统将在将来用于产生将与th 3/+动物杂交的嵌合体。我们相信，在这些病理条件下改变的基因的鉴定和表征对于揭示控制这些基本生物过程的功能性遗传相互作用是必不可少的。这些研究的目的是促进开发β地中海贫血的新的药理学和遗传学治疗方法。

DESCRIPTION (provided by applicant): Beta-thalassemia is a monogenic disorder that arises as a result of mutations in the beta-globin gene and is characterized by an extremely complex phenotype. Reduction of expression of the human beta-globin gene interferes with iron metabolism and leads to ineffective erythropoiesis, splenomegaly and osteopenia. Despite discoveries concerning the genetic abnormalities that lead to the development of beta-thalassemia, cellular processes that are perturbed in this disease remain unsolved. Our working hypothesis is that reduction of beta-globin expression triggers modification of the expression levels of genes that contribute to the beta-thalassemia phenotype. Thus we propose to isolate and characterize genes that are perturbed in tissues affected by this disorder by studying alterations in their levels of expression under normal and pathological conditions. Recently, we have established the first mouse model of adult lethal Cooley's anemia by engrafting fetal liver cells homozygous for a deletion in the mouse beta-globin genes (th3/th3) into irradiated wild-type (wt) recipient animals. These mice die within 60 days of engraftment due to a profound anemia resulting from ineffective erythropoiesis and massive iron overload. The development of this novel mouse model makes possible the identification of key genes involved in the pathophysiology of beta-thalassemia for the first time. As a first step we will compare (Aim 1) the gene expression profiles of tissues derived from the spleen, bone marrow and liver of wt animals, mice affected by beta-thalassemia intermedia and Cooley's anemia, using microarray analyses to identify differentially expressed genes. We predict that over-expression or inhibition of genes isolated through microarray analyses will reveal critical functional relationships between these genes and the beta-thalassemia syndrome. This will be tested in mice and, to modify the expression level of individual genes isolated through the microarray analyses, we will use a lentiviral vector system that we developed to repress or over-express target genes. We will utilize mice with the same genetic background from which the genes have been isolated in order to modify one parameter (the expression level of a single gene) at a time. Therefore, we will utilize a new lentiviral system to regulate, in a controlled fashion, expression of endogenous genes isolated by microarray analyses. In order to test (Aim 2) their function, in vivo, under normal and pathological conditions, embryonic stem (ES) cells will be transduced with this lentiviral system that will be used, in the future, to generate chimeras that will be intercrossed with th3/+ animals. We believe that the identification and characterization of genes that are altered under these pathological conditions is essential to reveal functional genetic interactions that govern these basic biological processes. These studies are intended to contribute to the development of new pharmacological and genetic therapeutic approaches for beta-thalassemia.