Efficient infection of primitive hematopoietic stem cells by modified adenovirus

Almost all studies of adenoviral vector-mediated gene transfer have made us e of the adenovirus type 5 (Ad5). Unfortunately, Ad5 has been ineffective a t infecting hematopoietic progenitor cells (HPC). Chimeric Ad5/F35 vectors that have been engineered to substitute the shorter-shafted fiber protein f rom Ad35 can efficiently infect committed hematopoietic cells and we now sh ow highly effective gene transfer to primitive progenitor subsets. An Ad5GF P and Ad5/F35GFP vector was added to CD34(+) and CD34(-)lineage- (lin(-)) H PC. Only 5-20% of CD34(+) and CD34(-) lin(-) cells expressed GFP after Ad5 exposure. In contrast, with the Ad5/F35 vector 30-70% of the CD34(+), 50-70 % of the CD34(-) lin(-) and up to 60% of the CD38(-) HPC expressed GFP and there was little evident cellular toxicity Because of these improved result s, we also analyzed the ability of Ad5/F35 virus to infect the Hoechst nega tive 'side population' (SP) of marrow cells, which appear to be among the v ery earliest multipotent HPC. Between 51% and 80% of marrow SP cells expres sed GFP. The infected populations retained their ability to form colonies i n two short-term culture systems, with no loss of viability. We also studie d the transfer and expression of immunomodulatory genes, CD40L (cell surfac e expression) and interleukin-2 (secreted). Both were expressed at immunomo dulatory levels for >5 days. The ability of Ad5/F35 to deliver transgenes t o primitive HPC with high efficiency and low toxicity in the absence of gro wth factors provides an improved means of studying the consequences of tran sient gene expression in these cells.