RETROVIRAL END-POINT TITER IS NOT PREDICTIVE OF GENE-TRANSFER EFFICIENCY - IMPLICATIONS FOR VECTOR PRODUCTION

Efforts to improve gene transfer (transduction) efficiency achieved wi th retroviral vectors often focus on increasing the end-point titer. I n this study, we assayed more than 70 retroviral vector supernatants f or end-point titer and for the ability to transfer reporter genes into cell populations (referred to as transduction efficiency). We found n o correlation between end-point titer and transduction efficiency. We also show that increasing end-point titer by ultrafiltration does not necessarily increase transduction efficiency. Evidence presented shows that nontransducing retroviral particles interfere with transducing v irions and reduce transduction efficiency without reducing end-point t iter. We have investigated production parameters and stability of retr oviral vector particles using transduction efficiency as a measure of supernatant potency. Analysis of the production kinetics showed that t he rate of virus production was marginally higher at 37 degrees C than at 32 degrees C. However, recombinant amphotropic retrovirus particle s are significantly more stable at 32 degrees C than at 37 degrees C. In addition, we show that short incubation periods are sufficient to y ield supernatants with high transduction efficiencies. We have impleme nted improved culture conditions, including short incubation periods, by continually perfusing medium over producer cells in a packed-bed bi oreactor incubated at 32 degrees C. By operating the packed-bed biorea ctor in perfusion mode, retroviral vector supernatants with a high tra nsduction efficiency can be routinely produced in large quantities.