Sickle cell anemia, a genetic disorder caused by a specific mutation in codon 6 of the human beta globin gene, results in severe morbidity and early mortality. None of the existing therapies have proved as effective as initially hoped. Currently, the development of gene transfer for sickle cell anemia has emerged as an important therapeutic option. Recent advances in 3 separate areas: l) identifying elements which confer high level, erythroid-specific globin gene expression, 2) isolating and culturing human pluripotent hematopoietic stem cells, and 3) developing new gene transfer technologies, ultimately may permit correction of the underlying genetic defect causing sickle cell anemia. Our overall objective is to adapt our existing cationic liposome-based gene delivery technology in order to develop safe and effective gene therapy for sickle cell anemia. To accomplish this, we will pursue, in parallel, both ex vivo and in vivo, liposome-based, gene transfer approaches. These two strategies are both separate and complementary. We will take advantage of assays provided by the stem cell core facilities and the availability of a transgenic sickle cell anemia mouse model, both of which are provided within this program project, to test the efficacy of our in vitro and in vivo gene transfer procedures, respectively. Each approach is based on the availability of the cationic liposome formulations, expression plasmid constructions, and DNA:lipid combinations that we have already shown can efficiently transfer and express gene constructs both in cultured cells, and in animals. (1) Our first goal is to identify conditions necessary to achieve liposome-based, efficient, stable transfection of bone marrow- derived human CD34+ stem cells ex vivo. (2) Our second goal is to maximize the amount and duration of human beta-like globin gene expression in erythroid progenitor cells of mice after cationic liposome-mediated in vivo gene delivery. To achieve these aims, we will optimize the cationic liposome formulation, the promoter and enhancer element used, design of the expression plasmid topology, DNA:lipid ratio, and targeting-ligands attached to the liposome surface, specifically for ex vivo and in vivo transfer and expression of human beta globin genes. We will measure the amounts of human beta globin mRNA and protein produced in these cells and determine the proportion of target cells that express the gene product. We will target uptake and expression of the beta globin gene to erythroid progenitor cells in mice, following systemic delivery of DNA:liposome complexes. We will focus on targeting the expression of these genes by linking them to beta globin promoter and enhancer sequences from the beta globin locus control region (LCR). These sequences have been shown to confer high level, erythroid-specific expression of linked human beta- globin genes. In summary, we will use our combined experience with cationic liposome-mediated gene delivery and the regulation of globin gene expression in order to A) stably transfect human beta-globin genes in human CD34+ stem cells ex vivo and B) to deliver and express these genes in murine erythroid progenitor cells for significant periods in vivo.
Showing the most recent 10 out of 25 publications
