Complexation of retrovirus with cationic and anionic polymers increases the efficiency of gene transfer

Previously, we have demonstrated that chondroitin sulfate proteoglycans and glycosaminoglycans inhibit retrovirus transduction. While studying the mec hanism of inhibition, we found that the combined addition of equal-weight c oncentrations (80 mug/ml) of Polybrene and chondroitin sulfate C to retrovi rus stocks resulted in the formation of a high-molecular-weight retrovirus- polymer complex that could be pelleted by low-speed centrifugation. The pel leted complex contained more than 80% of the virus particles, but less than 0.3% of the proteins that were originally present in the virus stock. Surp risingly, the virus in the complex remained active and could be used to tra nsduce cells. The titer of the pelleted virus, when resuspended in cell cul ture medium to the starting volume, was three-fold greater than the origina l virus stock. The selectivity (CFU/mg protein) of the process with respect to virus activity was more than 1000-fold. When the pelleted virus-polymer complex was resuspended in one-eighth of the original volume and used to t ransduce NIH 3T3 murine fibroblasts and primary human fibroblasts, gene tra nsfer was increased 10- to 20-fold over the original unconcentrated retrovi rus stock. The implications of our findings for the production, processing, and use of retrovirus stocks for human gene therapy protocols are discusse d.