Critical factors influencing stable transduction of human CD34(+) cells with HIV-1-derived lentiviral vectors

Lentiviral vectors have been proposed as a more efficient alternative to Mo loney murine leukemia virus-based retroviral vectors for transduction of hu man hematopoietic progenitors and stem cells. These studies were designed t o evaluate the conditions that influence transduction frequency of CD34(+) progenitors, with the goal of optimizing efficiency of stable gene transfer with lentiviral vectors. CD34(+) human cord blood cells and 293 cells were transduced with a human immunodeficiency virus (HIV)-1 derived lentiviral vector pseudotyped with vesicular stomatitis virus glycoprotein and carryin g an internal human cytomegalovirus promoter driving enhanced green fluores cent protein (eGFP) expression. Using fluorescence-activated cell sorting a nalysis of eGFP, we observed pseudotransduction beginning at the time of ve ctor addition and lasting up to 24 h in CD34(+) cells and up to 72 h in 293 cells. Integrase-defective lentiviral vector caused transient eGFP express ion for up to 10 days in CD34(+) cells and for up to 14 days in 293 cells. Protamine sulfate conferred no increase in transduction efficiency of CD34( +) cells on fibronectin-coated plates. Transduction frequency was related d irectly to vector concentration and not to multiplicity of infection across the ranges tested. First- and second-generation lentiviral vectors transdu ced CD34(+) cells equally, demonstrating a lack of dependence on HIV-1 acce ssory proteins. These findings will be useful for the optimal utilization o f this new class of vectors for transduction of human hematopoietic stem ce lls.