MEMBRANE ADSORPTION CHARACTERISTICS DETERMINE THE KINETICS OF FLOW-THROUGH TRANSDUCTIONS

Retrovirus-mediated gene transfer is currently limited by random Brown ian motion of the retrovirus. This limitation can be overcome by flowi ng the retrovirus solution through a porous membrane that supports the target cells, leading to a significant increase in the transduction e fficiency. This procedure is termed ''flow-through transduction.'' In this study, we characterized the effects of the fluid flowrate and the influence that membrane characteristics have on the flow-through tran sduction procedure. The transduction efficiencies increased with flowr ate until a plateau was reached at average flow velocities exceeding 0 .3 cm/h for flow times of 3 to 4 h, using a collagen-coated depth (COL ) membrane. A correlation between the optimal time for maximal gene tr ansfer using flow-through transductions and the optimal time for maxim al virus activity on the membrane was found, suggesting that the membr ane adsorption capacity for virus determined the amount of gene transf er that could occur. Membrane adsorption characteristics were further investigated using two different membrane types: a track-etched polyes ter screen (PE) membrane and the COL membrane. Flow-through transducti ons using the PE and COL membranes showed that a high level of gene tr ansfer could be attained using the COL membrane while the PE membrane gave much lower transduction efficiencies. The addition of the polycat ion polybrene (PB) changed these results markedly, making transduction s achieved on the PE membrane similar in number to those obtained on t he COL membrane. Since PB is believed to influence the virus adsorptio n to PE membrane, these results further support the conclusion that th e increase in gene transfer achieved by the flow-through transduction procedure is due to virus adsorption to the membrane. The flow-through transduction procedure thus leads to colocalization of the viral vect or and the target cell that in turn leads to a high transduction effic iency. (C) 1996 John Wiley & Sons, Inc.