Efficient gene transfer into @entity1583 stem cells offers a number of potential therapeutic applications. However, the relatively low titer of retroviral supernatants and the requirement for cell division to ensure integration have meant that transduction efficiency has been low. We have modified a flowthrough approach to cell transduction and have been able consistently to increase gene transfer efficiency into @entity1 @entity1583 progenitor cells. We transduced @entity2280 cells with retroviral vectors encoding a truncated @entity7169 ( @entity7169 ) or neo. Retroviral supernatant was pulled through 0.2-micron polycarbonated membranes, followed by placement of cells on the filter. In the absence of cytokines, the transduction efficiency of @entity2280 cells with a @entity7169 vector was increased 3-11-fold over that obtained at an identical MOI in liquid culture to produce 11%-44% transduction. Furthermore, both @entity5399 + and @entity5399 - subsets in a total @entity2280 population were transduced with similar efficiency, and transduction with a neo vector, as measured by G418 resistance in clonogenic assays, increased 1.5-5-fold. The mechanism by which gene transfer is improved may reflect colocalization of cells and retrovirus. Costaining of cells transduced on the filter with an @entity7169 retrovirus with both an @entity7169 antibody and a @entity12111 antibody that recognizes viral coat protein revealed high-level coexpression. The levels of in vitro gene transfer we obtain are equivalent to those observed when @entity2280 cells are cocultured in liquid culture with cytokines. However, culture with cytokines may commit @entity2280 cells to differentiation and has produced disappointingly low levels of subsequent in vivo gene transfer. Gene marking studies using distinguishable retroviral vectors will provide a means of learning whether the effects of flowthrough transduction genuinely enhance the efficiency of gene transfer to @entity1 marrow-repopulating cells.

Increased transduction efficiency of primary XXXX cells by physical colocalization of retrovirus and target cells.

@entity1 :: ('9606', 'Species') :: ['human'] @entity7169 :: ('4804', 'Gene') :: ['NGFR', 'nerve growth factor receptor'] @entity5399 :: ('7070', 'Gene') :: ['Thy1'] @entity12111 :: ('133418', 'Gene') :: ['gp70'] @entity1583 :: ('MESH:D019337', 'Disease') :: ['hematopoietic'] @entity2280 :: ('947', 'Gene') :: ['CD34']
@entity1583 :: (MESH:D019337,Disease) :: ['hematopoietic']
