Retroviral end-point titer is not predictive of gene transfer efficiency: implications for vector production.
Efforts to improve gene transfer (transduction) efficiency achieved with retroviral vectors often focus on increasing the end-point titer. In this study, we assayed more than 70 retroviral vector supernatants for end-point titer and for the ability to transfer reporter genes into cell populations (referred to as transduction efficiency). We found no 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 virions and reduce transduction efficiency without reducing end-point titer. We have investigated production parameters and stability of retroviral vector particles using transduction efficiency as a measure of supernatant potency. Analysis of the production kinetics showed that the rate of virus production was marginally higher at 37 degrees C than at 32 degrees C. However, recombinant amphotropic retrovirus particles are significantly more stable at 32 degrees C than at 37 degrees C. In addition, we show that short incubation periods are sufficient to yield supernatants with high transduction efficiencies. We have implemented improved culture conditions, including short incubation periods, by continually perfusing medium over producer cells in a packed-bed bioreactor incubated at 32 degrees C. By operating the packed-bed bioreactor in perfusion mode, retroviral vector supernatants with a high transduction efficiency can be routinely produced in large quantities.
Forestell
S P
SP
Progenesys, Palo Alto, California 94304, USA.
Böhnlein
E
E
Rigg
R J
RJ
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D019868:Kanamycin Kinase; D007700:Kinetics; D051379:Mice; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D011237:Predictive Value of Tests; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015203:Reproducibility of Results; D012190:Retroviridae; D013696:Temperature; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D001616:beta-Galactosidase