OPTIMIZATION OF FIBRONECTIN-ASSISTED RETROVIRAL GENE-TRANSFER INTO HUMAN CD34(-CELLS() HEMATOPOIETIC)

Efficient retroviral gene transfer into hematopoietic stem and progeni tor cells can be achieved by co-localizing retrovirus and target cells on specific adhesion domains of recombinant fibronectin (FN) fragment s, In this paper, we further optimize this technology for human CD34() cells, Investigating the role of cytokine prestimulation in retrovir us-mediated gene transfer on plates coated with the recombinant FN CH- 296 revealed that prestimulation of granulocyte colony-stimulating fac tor (G-CSF)-mobilized peripheral blood (PB) CD34(+) cells was essentia l to achieve efficient gene transfer into clonogenic cells, The highes t 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 CD 34(+) PB cells to retrovirus and cytokines in the presence of CH-296 l owered the gene transfer efficiency, Gene transfer into cytokine prest imulated CD34(+) bone marrow (BM) cells was not influenced by increasi ng the coating concentrations of a recombinant FN fragment, CH-296, no r was it adversely influenced by increasing the number of CD34(+) targ et cells, suggesting that the amount of retroviral particles present i n the supernatant was not a limiting factor for transduction of CD34() BM cells on CH-296-coated plates, The polycation Polybrene was not r equired for efficient transduction of hematopoietic cells in the prese nce 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 C H-296. These findings establish a simple and short clinically applicab le transduction protocol that targets up to 68% of BM or G-CSF-mobiliz ed PB CD34(+) cells and is capable of genetically modifying up to 17% of CD34(+)CD38(-)/dim PB cells.