Gene transduction of stem cells is a key requisite for successful gene therapy of a variety of genetic disorders of hematolymphoid cells, such as hemoglobinopathies, immune deficiencies and storage disorders. In contrast to the widely demonstrated ability to transduce high percentages of pluripotent murine stem cells, efforts with large animal models and preliminary human trials have frustratingly shown much lower extents of gene transfer into stem cells (1-2%) maximum). Thus, a key basic research goal must be to derive techniques to study gene transduction of human stem cells. We have developed a unique model system for the sustained growth of adult human bone marrow cells in vivo in immune deficient mice (beige/nude/xid = bnx). The system which we have developed requires only a single tail vein injection. We have demonstrated that sustained human hematopoiesis can be achieved from transplanted CD34+ progenitor cells if they are co- injected with human bone marrow stromal cells which have been engineered to secrete human IL-3. The presence of functional human multilineage colony-forming progenitor cells and mature myeloid cells was demonstrated in the marrow, spleens and blood of the mice for up to eleven months. When this model of human hematopoiesis was used to study gene transfer into human hematopoietic cells using retroviral vectors, the presence and function of the inserted gene was consistently demonstrated in human colony-forming progenitor cells isolated from the mice after 4-11 months. The major limitation of the system to date is that we have not detected human B lymphoid cells produced in the mice. Our central hypothesis is that human hematopoietic stem cells will undergo pluripotent differentiation (lymphoid as well as myeloid) in immune deficient mice if provided with appropriate human growth factors by co-transplantation of engineered stromal cells. Pluripotent hematopoiesis will permit us to analyze and optimize gene transfer into human stem cells. To achieve this goal we will transplant human CD34+ cells (and, subsequently, subsets such as CD34+, CD38-) into immune deficient mice along with human marrow stromal cells engineered to produce human IL-2, IL-7, LIF, Stem Cell Factor (SCF), flt-3 ligand and/or IL-3. We will then analyze the extent of engraftment of human hematopoietic cells and compare the types of mature cells produced, specifically seeking the production of lymphoid cells in addition to myeloid cells. We will then use this system to study methods to achieve maximal gene transduction of human pluripotent stem cells. We will also use this system to compare adeno-associated virus (AAV) vectors to retroviral vectors. The presence of the same vectors integrant in cells of multiple lineages will demonstrate stem cell transduction. The goal of this portion of the proposal is to develop protocols which would have direct clinical applications for gene therapy of genetic diseases of hematopoietic and lymphoid cells.
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