Optimized retroviral transduction protocol for human progenitor cells utilizing fibronectin fragments

Background Retroviral transduction in the presence of fibronectin (FN) fragments has p roven an efficient and clinically-applicable procedure for gene transfer in to hematopoietic cells. So far, FN-based transduction protocols have been o ptimized primarily for transduction of stem cells, whereas for several ther apeutic applications transduction of clonogenic progenitors (CFU) may be su fficient. Methods Transduction protocols for CFU were optimized by evaluating the effect of g rowth factors, timing of retroviral transduction, CD34-selection and hepari n, using a neomycin-phosphotransferase (neo(R))-expressing retroviral vecto r. Results The presence of multiple growth factors during prestimulation and transduct ion, including the differentiating cytokines G-CSF or GM-CSF, substantially enhanced transduction of CFU. Best results were achieved when 24 h of pres timulation were followed by a 24-48 h transduction period of the presence o f the CH-296 FN-fragment and IL-3, IL-11, SCF, erythropoietin (EPO), and GM -CSF. With this protocol we observed highly efficient transduction of BM-de rived CFU (90.7 +/- 8.8% G 418-resistant colonies), even with retrovirus pr eparations of moderate infectious titer (5 x 10(4) - 2 x 10(5) CFU/mL). The number of CFU increased on average 2.6-fold (range 1.5-3.8) during the tra nsduction procedure. Selection of CD34(+) cells prior to transduction did n ot improve transduction efficiency. Heparin, even in concentrations as low as 2.0 mug/mL, significantly inhibited transduction of CFU on FN-fragments. Discussion An optimized protocol for retroviral gene transfer into human clonogenic pr ogenitor cells that allows highly efficient transduction, even with moderat e titer retroviral vectors, is presented.