Retroviral vector production by transient transfection of 293VEC-GALV cells

Background & Aim Introduction Retroviral vectors can permanently integrate their genetic material into chromosomes in the target cells for long-term, stable expression of the introduced genetic elements. It has been widely used since the 1980s and is the second most commonly employed system for gene transfer. Traditionally, retroviral vector production involves the preparation and characterization of a stable producer cell line from which the vector is produced as supernatant. It usually takes about 12 to 20 months from generation the stable producer cell line clone to the final release of the retroviral vector for clinical use. Due to the recent rapid growth of CAR-T cell therapies, a faster, more efficient method for retroviral vector production is required. We have, therefore, employed a novel manufacturing system using transient transfection for rapid GMP preparation of retroviral vectors. Methods, Results & Conclusion Methods Clinical retroviral vectors are produced from a master cell bank of 293Vec-Galv (BioVec Pharma, Quebec, Canada), a human embryonic kidney HEK293-based packaging cell line that stably expresses the Moloney Murine Leukemia gag-pol and Gibbon Ape Leukemia Virus envelope viral proteins. The cells are transfected with plasmid DNA that carries the gene(s) of interest (purified plasmid DNAs are produced by Puresyn Inc. Malvern PA, USA or Aldevron Inc. Fargo ND, USA). The transfection is carried out at 37°C and is mediated by PEIpro®-HQ (Polyplus Transfection, Illkirch, France), which is a highly qualified grade of polyethylenimine developed for large-scale bioproduction processes. About 24 hours post-transfection the medium is replaced by fresh complete medium. Supernatant containing the retrovirus is harvested at 48, 72 and 96 hours post-transfection and filtered through 0.45µm filter. The filtered supernatant is aliquoted, snap frozen and immediately transferred to storage in -80°C freezers. Results and Conclusion Eight retroviral vectors have been manufactured using this novel system at the CAGT GMP Vector Production Facility. Vector production from a single master cell bank of 293Vec-Galv takes about 4 weeks. Four vectors have been released for clinical trials. The transduction rate, phenotype and cytotoxic effect of T cells transduced by vectors produced by this novel system met release requirements, including absence of replication-competent retrovirus. Retroviral vector production by transient transfection of 293Vec-Galv cells saves time, is cost-efficient and safe ( Table 1 ).