ENHANCEMENT OF ADENOVIRUS-MEDIATED GENE-TRANSFER TO HUMAN BONE-MARROWCELLS

Adenovirus infection of CD34(+) hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or abse nce of growth factors. Studies revealed that a brief co-incubation (1- 8 hours), resulted in low levels of transgene expression, suggesting t hat adenovirus infection of CD34(+) cells occurs slowly, and optimal t ransduction requires a 24 hour exposure to adenovirus. Infection by Ad /beta-gal or Ad/p53 at a MOI of 500:1 provided a high transduction eff iciency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 inf ected stem cells. We conclude that a 24 hour exposure to recombinant a denovirus encoding p53 or beta-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on he matopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with imp roved gene delivery and maintenance of hematopoietic function. In summ ary, adenovirus vectors can be used to transiently transduce stem cell s, and conditions have been defined to maximize expression and limit i nhibitory effects on CD34(+) cells. These data support continued inves tigation of this vector for local cytokine delivery and purging of ste m cell products.