Despite intensive efforts to develop effective therapy for sickle cell anemia (SCA), this disease continues to be associated with significant morbidity and mortality. SCA affects 0.2% of African American children and young adults. In order for future gene therapy-based strategies for SCA to be successful: 1) transduction of self-renewing stem cells must be highly efficient, 2) transduced cells must have a selective or proliferative advantage, and 3) gene delivery vectors must produce stable, therapeutic levels of globin gene expression over the lifetime of the individual. Our goal is to develop procedures for efficient gene transfer into fetal liver and have already shown that high-level gene expression may be achieved following either intraperitoneal or direct intrahepatic injection of viral or non-viral vectors. We will focus on the transduction of highly proliferative HSC in the murine fetal liver. Our first hypothesis is that direct in utero delivery of gene transfer vectors will result in the transduction of higher numbers of HSCs than can be achieved in vitro, and without disrupting either the microenvironment or biology of these early HSC. We will determine the most efficient vector system for gene transfer into totipotent fetal HSC using MLV- and HIV-based retroviral vectors, and adeno-associated viral vectors. We will focus on the transduction of highly proliferative HSC in the murine fetal liver. Our first hypothesis is that direct in utero delivery of gene transfer vectors will result in the transduction of higher numbers of HSCs than can be achieved in vitro, and without disrupting either the microenvironment or biology of these early HSC. We will determine the most efficient vector system for gene transfer into totipotent fetal HSCs using MLV- and HIV- based retroviral vectors, and adeno-associated viral vectors. Our second hypothesis is that transuterine injection provides an efficient model for rapidly screening novel globin gene vectors. We will deliver human gamma or beta gene expression. The therapeutic efficacy of gamma or beta globin vectors that direct high-level expression of globin will be tested in murine models of beta thalassemia and sickle cell anemia. Vectors that produce high-level globin gene expression will reduce red cell sickling and confer a survival advantage of transduced red cells. These studies will also define the fate of transduced hematopoietic stem cells and their progeny during ontogeny.
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