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.
