High efficiencies of gene transfer with immobilized recombinant retrovirus: Kinetics and optimization

We used a combination of mathematical modeling and experiments to investiga te the rate-limiting steps of retroviral transduction. on surface-bound fib ronectin (FN) and identify the conditions that maximize the efficiency of g ene transfer. Our results show that fibronectin-assisted gene transfer (FAG T) is a strong function of the time and temperature of virus incubation in FN-coated plates. Gene transfer increases sharply at short times, reaches a maximum at intermediate times, and eventually declines as a result of loss of retroviral activity. The maximum transduction efficiency and the time a t which this is attained increase with decreasing temperature of virus incu bation. Depending on the temperature and the type of target cells, the init ial rate of gene transfer increases by 3- to 10-fold and the maximum transd uction efficiency increases by 2- to 4-fold as compared to traditional tran sduction (TT). Interestingly, Polybrene (PB) inhibits FAGT in a dose-depend ent manner by inhibiting binding of retrovirus to FN. In contrast to tradit ional transduction, FAGT yields higher than 10-fold transduction efficienci es with concentrated retrovirus stocks. Gene transfer is directly proportio nal to the concentration of the virus-containing medium with no sign of sat uration for the range of concentrations tested. These results suggest that immobilization of recombinant retrovirus can be rationally optimized to yie ld high efficiency of gene transfer to primary cells and improve the prospe ct of gene therapy for the treatment of human disease.