Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells

Efficient retroviral gene transfer into hematopoietic stem and progenitor cells can be achieved by co-localizing retrovirus and target cells on specific adhesion domains of recombinant fibronectin (FN) fragments, In this paper, we further optimize this technology for human CD34(+) cells, Investigating the role of cytokine prestimulation in retrovirus-mediated gene transfer on plates coated with the recombinant FN CH-296 revealed that prestimulation of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) CD34(+) cells was essential to achieve efficient gene transfer into clonogenic cells, The highest gene transfer occurred by prestimulating PB CD34(+) cells for 40 hr with a combination of stem cell factor (SCF), G-CSF, and megakaryocyte growth and development factor (MGDF) prior to retroviral infection on CH-296. Surprisingly, a prolonged simultaneous exposure of primary CD34(+) PB cells to retrovirus and cytokines in the presence of CH-296 lowered the gene transfer efficiency, Gene transfer into cytokine prestimulated CD34(+) bone marrow (BM) cells was not influenced by increasing the coating concentrations of a recombinant FN fragment, CH-296, nor was it adversely influenced by increasing the number of CD34(+) target cells, suggesting that the amount of retroviral particles present in the supernatant was not a limiting factor for transduction of CD34(+) BM cells on CH-296-coated plates, The polycation Polybrene was not required for efficient transduction of hematopoietic cells in the presence of CH-296, Furthermore, we demonstrated that repeated exposure of CH-296 to retrovirus containing supernatant, called preloading, can be employed to concentrate the amount of retroviral particles bound to CH-296. These findings establish a simple and short clinically applicable transduction protocol that targets up to 68% of BM or G-CSF-mobilized PB CD34(+) cells and is capable of genetically modifying up to 17% of CD34(+)CD38(-)/dim PB cells.