Increased transduction effciency of primary hemopoietic cells by physical co-localization of rethovirus and target cells

Efficient gene transfer into hemopoietic stem cells offers a number of therapeutic applications. However the relatively low titre of retroviral supernatants and the requirement for cell division to ensure integration has meant that transduction efficiency has been low. We have modified a "flow through" approach to cell transduction (Human Gene Therapy 7:743, 1996) and have been able to increase gene transfer efficiency into human hemopoietic progenitor cells. We transduced cells with retroviral vectors encoding a truncated nerve growth factor receptor (NGFR) or neo. Retroviral supernatant was pulled through 0.2 æ polycarbonated membranes followed by placement of cells on the filter. In the absence of cytokines, the transduction efficiency of CD34 cells with a NGFR vector was increased 3-11 fold over that obtained at an identical MOI in liquid culture to produce 11-44% transduction. Furthermore both Thy1+ and Thy1- subsets were transduced with similar efficiency. Similarly transduction with a neo vector as measured by G418 resistance in clonogenic assays increased 1.5 to 5 fold. The mechanism by which gene transfer is improved may reflect co-localization of cells and retrovirus. Costaining of cells transduced on the filter with an NGFR retrovirus with both an NGFR antibody and a gp70 antibody that recognizes viral coat protein revealed high level co-expression. These levels of invitro gene transfer are equivalent to those observed when CD34 cells are cocultured with cytokines. Gene marking studies using distinguishable retroviral vectors will provide a means of learning the effects of flow though transduction on the efficiency of gene transfer to human marrow repopulating cells.