HEMOPOIETIC STEM CELL SELF RENEWAL AND RETINOID ANTAGONISTS

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Progress in utilizing retroviral vectors to correct hereditary disorders involving the hematopoietic stem cell (HSC) has been exceedingly slow. One of the major stumbling blocks relates to the observation that retroviral vectors preferentially infect mitotically active cells, but the normally quiescent HSC, when stimulated to divide in vitro, frequently commits itself to differentiate into a more mature, lineage committed stem cell exhibiting a limited clonal lifespan. It is these lineage committed proliferating progenitors exhibiting both a limited lifespan and poor long-term marrow repopulating ability that are preferentially infected by the retroviral vectors. Thus the successful use of retroviral vectors to correct HSC genetic disorders requires that self renewing HSC be preferentially expanded and transduced by these vectors. We wish to determine the specific culture conditions that will promote the self- renewal of HSC in vitro and will lead to more efficient retroviral vector mediated transduction of HSC. Our experimental approach will be based on recent observations in our laboratory indicating that blocking the activity of retinoic acid (RA) receptors in hematopoietic stem cells inhibits their lineage commitment and enhances their self renewal. Our specific goals are as follows: SPECIFIC AIM I) Determine the in vivo marrow repopulating capability of the cultured murine lymphohematopoietic EML cells. We wish to determine the capacity of primitive SCF-dependent mouse cell lines (designated EML) derived by transducing normal mouse bone marrow with a dominant negative RA receptor construct, to function in vivo as stem cells in irradiated syngeneic or in SCID mice. SPECIFIC AIM II) Determine the optimal in vitro conditions that enhance the self renewal of murine and human hematopoietic stem cells. Highly enriched fractions of murine and human HSCs will be cultured in vitro under various conditions with hematopoietic stem cell growth factors together with synthetic retinoids exhibiting RA receptor antagonism. The self renewal of HSCs in these cultures will be evaluated by various techniques. SPECIFIC AIM III) Determine in vitro conditions for optimizing retroviral mediated gene transduction into human hematopoietic stem cells. Utilizing culture conditions determined in Specific Aim Il we will infect self renewing hematopoietic stem cells with retroviral vectors harboring specific markers and assess the efficiency of successfully transducing these HSCs with these vectors. These studies directly address the problem related to inefficient retroviral vector mediated gene transduction into hematopoietic stem cells. Our approach, if successful, will have broad applicability to gene therapy of patients with a variety of different genetic disorders of hematopoietic stem cells.