Sickle cell anemia (SCA), a genetic disorder affecting 0.2% of American blacks, results in severe morbidity and early mortality. Although the specific mutation that causes this disease was described more that thirty years ago, no clearly effective therapy for SCA exists. Recently, dramatic advances have been made in gene targeting and gene transfer techniques, as well as in understanding the mechanisms regulating human gene expression. Manipulation of the human beta-globin locus has now become a plausible therapeutic option for SCA, but will require: 1) reliable animal models, 2) efficient techniques for gene targeting and gene transfer in mammalian stem cells, and 3) a comprehensive understanding of the mechanisms that control tissue-and developmental stage-specific expression of the human beta-globin gene family. Our goal is to more precisely identify the regulatory sequences in the beta-globin locus and to define their functional role during ontogeny. It has not been possible to study the physiological role of cis-acting sequences within the intact human beta-globin region (90 kb) due to the insert size limitations of plasmid (10 kb) and cosmid (40 kb) vectors. However, the recently developed method for cloning genomic fragments of several hundred kb or more into yeast artificial chromosomes (YACs), has now made it feasible to isolate large genetic loci such as the B-globin locus. We will combine the advantages of YAC technology with gene transfer methods, in order to introduce YACs containing the beta-globin gene locus into murine cell lines and transgenic mice. We have extensively characterized two YACs containing the beta-globin gene family including 5' and 3' flanking regions in a single contiguous insert. Using these YACs, we will adapt methods for reliably introducing high molecular weight DNA into cells, while preserving the genomic structure of the fragments after their integration into host chromatin. We will assess the relative advantages of different transfection procedures using murine erythroleukemia cells or embryonic stem (ES) cells, or the microinjection of oocytes for this purpose. Once we have produced cell lines carrying the entire human Beta-globin gene locus, we will use quantitative RNA analysis to study the expression of members of the gene family. We will use homology-directed recombination in yeast to generate mutations of potential regulatory regions. YACs containing these mutations will be transferred into mice or cell lines using the procedures we have developed. The effect of these mutations on tissue and developmental stage specific expression will be analyzed. We believe this work will provide a model system for better understanding the regulation of the human beta-globin locus during ontogeny. Furthermore, the development of transgenic mice and ES cell lines carrying the entire human beta- globin gene locus will allow pre-clinical testing of new pharmacologic agents as well as of gene transfer and targeting procedures, as potential treatments for SCA.
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