Retroviral transduction of human CD34(+) cells on fibronectin fragment CH-296 is inhibited by high concentrations of vector containing medium

Background The objective of the present study was to optimize conditions fo r retroviral transduction of human cord blood (CB) CD34(+) cells and to rev eal mechanisms which interfere with efficient gene transfer. Methods An MSCV based retroviral vector with the gene for enhanced green fl uorescent protein (MGIN) produced by GP + envAM12 Camphotropic envelope), P G13 (gibbon ape leukemia virus envelope) or 293GPG (vesicular stomatitis vi rus envelope) cell lines was used to transduce cord blood CD34(+) cells on Retronectin (fibronectin fragment CH-296) in three different ways: either i n vector containing medium (VCM), in fresh medium on Retronectin pre-loaded with vector or in VCM on Retronectin pre-loaded with vector. Results Paradoxically, the transduction efficiency obtained with pre-load o f vector onto Retronectin alone was higher than pre-load plus VCM for PG13- MGIN (67.9 +/- 6.0% vs 24.9 +/- 8.0%) and AM12-MGIN C47.5 +/- 5.8% vs 38.7 +/- 2.2%). Further experiments showed that transduction on Retronectin pre- loaded with PG13-MGIN or AM12-MGIN was inhibited by the presence of the sam e VCM at high concentrations, but not by the presence of a VCM with a diffe rent receptor specificity. If no pre-load of vector was performed, the high est transduction efficiencies were seen when VCMs were diluted 1:10 (MOIs o f 3). The inhibitory effect of high titer PG13-MGIN VCM was confirmed in mo re primitive CD34(+)CD38(low) cells and in NOD/SCID repopulating cells, and was also seen in experiments with bone marrow CD34(+) cells. Conclusions Retroviral transduction of CB CD34+ cells on Retronectin is inh ibited by high titer PG13 and GP +envAM12 vector containing medium. Efficie nt gene transfer to human hematopoietic cells can be obtained by preload al one of the vector onto Retronectin. These findings are of importance for th e design of transduction protocols for repopulating hematopoietic cells. Co pyright (C) 2001 John Wiley & Sons, Ltd.