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

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Abstract
Background The objective of the present study was to optimize conditions for retroviral transduction of human cord blood (CB) CD34+ cells and to reveal mechanisms which interfere with efficient gene transfer. Methods An MSCV based retroviral vector with the gene for enhanced green fluorescent protein (MGIN) produced by GP+envAM12 (amphotropic envelope), PG13 (gibbon ape leukemia virus envelope) or 293GPG (vesicular stomatitis virus envelope) cell lines was used to transduce cord blood CD34+ cells on Retronectin (fibronectin fragment CH-296) in three different ways: either in 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 ofvector 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 (47.5±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 ofthe same VCM at high concentrations, but not by the presence of a VCM with a different receptor specificity. If no pre-load of vector was performed, the highest transduction efficiencies were seen when VCMs were diluted 1:10 (MOIs of 3). The inhibitory effect of high titer PG13-MGIN VCM wasconfirmed in more primitive CD34+CD38low 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 inhibited by high titer PG13 and GP+envAM12 vector containing medium. Efficient gene transfer to human hematopoietic cells can be obtained by pre-load alone of the vector onto Retronectin. These findings are of importance for the design of transduction protocols for repopulating hematopoietic cells. Copyright © 2001 John Wiley & Sons, Ltd.

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