Improved Methods of Retroviral Vector Transduction and Production for Gene Therapy

Abstract

To facilitate clinical applications of retroviral-mediated human gene transfer, retroviral vectors must be of high titer and free of detectable 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°C than at 32°C. A 5- to 15-fold increase of vectors was produced at 32°C compared to 37°C; the vector increase at 34°C was intermediate. For example, PA317/G1Na.40 grew to a titer of 1.8 × 107 cfu/ml at 32°C, compared to 5.0 × 105 cfu/ml at 37°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 combination of glucose or sorbitol with gelatin resulted in recovery rates of 64–83%. In studies on transduction by retroviral vectors, centrifugation of vector supernatants onto target cells significantly increased transduction efficiency as measured by vector titration for G418 resistance, fluorescence-activated cell sorting (FACS), and polymerase chain reaction (PCR) analyses. The combination of the above methods has significantly increased the growth and transduction by this vector system. For expanded clinical trials using retroviral-mediated gene transfer, large volumes of vector will be needed at the highest possible titers. Factors that influence large-scale production of retroviral vector need to be defined. The presentation of Kotani et al. identifies some factors that influence growth, concentration, transduction, and freeze drying of these vectors.