The long term goal of these studies is to develop human hematopoietic stem cell (hsc) gene replacement therapy. The ultimate goal is to develop a hsc gene replacement therapy of the disease leukocyte adhesion deficiency (LAD). LAD is a lethal disease caused by a defective CD18 gene. The specific objectives of these studies is to develop a murine hsc gene replacement therapy model that will have direct applicability to human hsc gene replacement therapy. This will be accomplished by thoroughly characterizing an existing murine hsc gene replacement therapy model and adapting it to make it more applicable to human hsc gene therapy. The current murine model relies on cocultivation of a human CD18 encoding ecotropic retrovirus cell lines with murine bone marrow and infusing it into lethally irradiated,syngeneic recipient mice. This model will be adapted such that human CD18 encoding amphotropic retrovirus supernatants will be used to transduce bone marrow before bone marrow transplantation. The murine model will be thoroughly characterized by several means. First, hsc frequency in the infused, genetically modified marrow will be assessed by performing limiting dilutions of bone marrow cells into lethally irradiated mice and assaying for survival of the mice and engraftment of the infused marrow. Hsc engraftment will be analyzed by assaying for the persistance of Y chromosome specific sequences in hematopoietic tissues of male mice transplanted with female bone marrow. Second, gene transfer into the hsc will be analyzed by assessing the hematopoietic tissues for the presence of intact proviral sequences. Third, transgene expression will be analyzed by assessing the hematopoietic tissues for the presence of provirus derived RNA, and by analyzing for hematopoietic cell surface human CD18 expression. After achieving successful gene transfer and expression with amphotropic virus supernatants, the effect of various hematopoietic growth factors on hsc survival, hsc engraftment, gene transfer into the hsc, and gene expression in cells derived from the hsc will be thoroughly studied. The immunophenotype of the transgene (human CD18) expressing and non-expressing cells will be characterized by performing two color flow cytometry. In addition, the use of bone marrow cells enriched for hsc's versus unfractionated bone marrow as the target cells for gene transfer will be assessed. Finally, development of an in vitro assay that is predictive of successful gene transfer into the hsc will be undertaken. If these studies are successful, they will provide for a thorough understanding of the biologic factors important in successful hsc gene transfer. This will facilitate the development of human hsc gene replacement therapy.
