title: Evaluation of safety and methods for production of retroviral vectors for use in gene therapy
Creators: name: Kewney, Caroline
Publisher: name: University of Glasgow
Year: 1998

description: Retroviral vectors are replication-defective viral genomes, in which genes essential for viral replication have been replaced by therapeutic genes. Essential genes are provided in trans by a helper virus, or packaging cell line. Stable producer cell lines are selected following transfection or infection of the vector into a packaging cell line. Such cell lines produce infectious, but replication incompetent virus particles containing the therapeutic gene(s) of interest. A potential hazard in the adaptation of retroviruses for use as gene delivery vehicles is the potential for recombination between vector and packaging cell line sequences to produce replication-competent retrovirus (RCR). The primary objective of the present study was to validate and determine optimal conditions for assays employed in the detection of RCR. The results indicated that murine Mas dunni and feline PG4 S+L- cells were of similar sensitivity to infection by amphotropic murine leukaemia virus (A-MLV); three passages were found to be sufficient to detect the presence of RCR at a virus input of 10-1 infectious units/ml. Using co-cultivation procedures it was possible to detect 1 RCR in a total of 107cells. It was determined that the presence of replication incompetent vector reduced the sensitivity of direct PG4 S+L- assay to detect wild-type A-MLV. This effect was reduced in extended Mus dunni and PG4 S+L- assays while the presence of retroviral vector producing cells did not affect the detection of A-MLV in co-cultivation assays. Accordingly, it is recommended that co-cultivation and/or extended assays should be performed for detection of RCR. In addition, studies were performed to evaluate the parameters which influence production of a selected retroviral vector, including determination of optimal cell culture and harvesting conditions, investigating scale-up of production, and assessing stability and methods of concentrating viral vector supernatants. Parameters for optimal cell growth coupled with maximum viral titre for a model producer cell line were defined using tissue culture flasks, and found to be suitable for scale-up without loss of viral titre into roller bottles or Fibra-Cel disks in a spinner culture flask. Producer cells could also be adapted to culture in serum-free media although cell expansion was slower and a decrease of approximately 1 log10 unit in viral titre was observed. Ultrafiltration was assessed as a method for increasing viral titre, where it was demonstrated that viral titre could be increased by 1 LOG10 unit following concentration. The stability of vector supernatant was examined by assessing viral titre following storage at different temperatures in relation to wild-type A-MLV. Resulting viral titres indicated that supernatant could be stored at -80°C for six months without loss of titre, whereas at 4°C titre decreased within 5 days. At 37°C the half-life of viral vector was determined to be approximately 5 hours after which complete inactivation was observed. Although inactivation may have been expected to be complete within a short incubation period at 56°C, the results indicated that a fraction of the virus was resistant to inactivation following incubation for two hours.