Efficient transduction of human lymphocytes and CD34(+) cells via human immunodeficiency virus-based gene transfer vectors

The development of gene transfer systems for the efficient transduction of human primary cells including lymphocytes and CD34(+) cells is a significan t step in the advancement of gene therapy and cell marking protocols. Effic ient gene transfer systems also represent useful tools for basic research. Here we show that human primary lymphocytes and CD34(+) cells can be effici ently transduced using a VSV-G pseudotyped HIV-l-based gene transfer system . The enhanced green fluorescent protein (EGFP) was chosen as the marker tr ansgene, because it can be easily visualized and quantitated using fluoresc ence microscopy and flow cytometry, thus eliminating the need for selection or PCR to score transduction. Vectors produced with this system did not ge nerate replication-competent retroviruses (RCRs) and efficiently transduced human cell lines (40-90%), PBMCs (60 %), mobilized CD34(+) cells (39%), an d CD34(+) cells from umbilical cord blood (60%) as measured by flow cytomet ry. Cells treated with AZT prior to infection did not express EGFP, ruling out passive protein or plasmid DNA transfer. This was further confirmed in methylcellulose cultures, where expression in myeloid and erythroid colonie s was maintained for at least 3 weeks. In addition, this HIV-based vector w as able to efficiently transduce freshly isolated, not-prestimulated CD34cells (70% EGFP positive) in serum-free medium. Under these same conditions , a Moloney murine leukemia virus-based vector failed to transduce not-pres timulated CD34+ cells. These characteristics make this gene transfer system an excellent choice for both basic science and possible gene therapy appli cations.