The major limitation to the use of gene therapy techniques in the hematopoietic system is the inability to efficiently transfer genes into hematopoietic stem cells of humans by using currently available gene transfer systems. However, it is clear that retroviral vectors are capable of transducing genes into hematopoietic stem cells in mice where the efficiency can approach 100%. The ability of a retrovirus to enter a cell is primarily determined by the binding of the retrovirus envelope protein to specific cell surface receptors that mediate virus entry. Thus, a better understanding of the receptors used by different retroviruses and their expression patterns in hematopoietic cells is important for improvement of hematopoietic stem cell transduction efficiency. We have previously cloned the murine amphotropic retroviral receptor and determined that it functions as a sodium-dependent phosphate transporter. We now wish to extend these studies to better characterize retroviral receptors on hematopoietic stem cells. Specifically we will first clone the MCF and xenotropic murine retrovirus receptors. With the already cloned ecotropic and amphotropic receptors, this will complete the murine retrovirus receptor group. We also plan to clone the receptor for the RD114 endogenous cat retrovirus, and may attempt to clone other receptors for primate retroviruses depending on our progress. Second we will study the relationship between receptor expression and gene transduction rates. Third, we will examine the expression of already cloned and newly cloned retrovirus receptors in CD34+ hematopoietic cells and further purified stem cells. Fourth, we will construct packaging cell lines with new receptor specificities based on results of the receptor studies in an attempt to increase infection efficiency in hematopoietic cells. Fifth, these studies will also investigate potential factors in hematopoietic cell cultures that may inhibit retrovirus infection. Overall, these studies will help determine the optimal vectors and most efficient methods for transduction of hematopoietic stem cells for the in vivo approaches in both non-human primates (Project 6) and humans (Project 7).
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