Optimized retroviral transduction protocol for human progenitor cells utilizing fibronectin fragments.

Abstract

Retroviral transduction in the presence of fibronectin (FN) fragments has proven an efficient and clinically-applicable procedure for gene transfer into hematopoietic cells. So far, FN-based transduction protocols have been optimized primarily for transduction of stem cells, whereas for several therapeutic applications transduction of clonogenic progenitors (CFU) may be sufficient. Transduction protocols for CFU were optimized by evaluating the effect of growth factors, timing of retroviral transduction, CD34-selection and heparin, using a neomycin-phosphotransferase (neo(R))-expressing retroviral vector. The presence of multiple growth factors during prestimulation and transduction, including the differentiating cytokines G-CSF or GM-CSF, substantially enhanced transduction of CFU. Best results were achieved when 24 h of prestimulation were followed by a 24-48 h transduction period in the presence of the CH-296 FN-fragment and IL-3, IL-11, SCF, erythropoietin (EPO), and GM-CSF. With this proto-col we observed highly efficient transduction of BM-derived CFU (90.7 +/- 8.8 % G 418-resistant colonies), even with retrovirus preparations 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 transduction procedure. Selection of CD34(+) cells prior to transduction did not improve transduction efficiency. Heparin, even in concentrations as low as 2.0 microg/mL, significantly inhibited transduction of CFU on FN-fragments. An optimized protocol for retroviral gene transfer into human clonogenic progenitor cells that allows highly efficient transduction, even with moderate titer retroviral vectors, is presented.