A variety of inherited and acquired human diseases affecting the hematopoietic stem cell (HSC) can potentially be treated with genetic manipulation of the patients HSCs. Potential applications of this technology include therapeutic approaches for common blood disorders (e.g. thalassemia), malignancies (e.g. leukemia) and infectious diseases of the blood cells, such as HIV-1 infection (AIDS). However, with a few exceptions, gene transfer into HSCs of large animal models and humans has thus far proven to be difficult and inefficient. By improving our knowledge of the biological characteristics of the human HSC we will likely gain important insights that will allow us to improve the efficiency of gene transfer in and correction of this elusive target cell. Under this project we have established in vitro and in vivo animal models of human hematopoiesis using hematopoietic progenitors obtained from cord blood, bone marrow or peripheral blood of volunteer donors. These cells are cultured in vitro in a variety of cytokines and growth factors to identify conditions that allow their survival in the absence of differentiation. In addition, the gene expression profile of these cells is studied by microarray analysis. The cells are then subjected to gene transfer using viral vectors that allow for integration of the transferred gene and allowed to differentiate into mature cell lineages in vitro (methylcellulose progenitor colonies) or in vivo using mouse or sheep animal models. The pattern of viral integration sites is studied to verify if trends indicating preferential genomic locations can be identified. These experiments allow comparison of cells with putative hematopoietic stem cell activity isolated from various sources (e.g. cord blood vs. bone marrow vs. peripheral blood) and testing of vectors and gene transfer conditions that efficiently target these cells. We have also developed an in vitro model of retroviral insertional oncogenesis that can be applied to murine and human hematopoietic stem cells to study the effects of genetic manipulation with retroviral vectors. These experiments will also allow to test the efficacy of safety modification of gene transfer vector that can be transferred to future human trials. We are employing strategies for precise editing of genome information to be used as correction tools for genetic diseases. These strategies will be applied to HSC obtained directly from mice and humans or through reprogramming technologies to test their safety and efficacy.
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