IMPROVED METHODS OF RETROVIRAL VECTOR TRANSDUCTION AND PRODUCTION FORGENE-THERAPY

To facilitate clinical applications of retroviral-mediated human gene transfer, retroviral vectors must be of high titer and free of detecta ble replication-competent retroviruses. The purpose of this study was to optimize methods of retroviral vector production and transduction. Studies were conducted using 22 retroviral vector producer cell lines. Inactivation of retroviral vectors was greater at 37 degrees C than a t 32 degrees C. A 5- to 15-fold increase of vectors was produced at 32 degrees C compared to 37 degrees C; the vector increase at 34 degrees C was intermediate. For example, PA317/G1Na.40 grew to a titer of 1.8 x 10(7) cfu/ml at 32 degrees C, compared to 5.0 x 10(5) cfu/ml at 37 degrees C. The production of retroviral vectors was scalable achieving similar results in flasks, roller bottles, or a CellCube Bioreactor. Retroviral vectors were concentrated 15-24 times with vector recovery ranging from 91 to 96% in a Pellicon tangential flow filtration system . Retroviral supernatants were successfully lyophilized, The combinati on of glucose or sorbitol with gelatin resulted in recovery rates of 6 4-83%. In studies on transduction by retroviral vectors, centrifugatio n of vector supernatants onto target cells significantly increased tra nsduction efficiency as measured by vector titration for G418 resistan ce, fluorescence-activated cell sorting (FACS), and polymerase chain r eaction (PCR) analyses. The combination of the above methods has signi ficantly increased the growth and transduction by this vector system.