473. Semliki Based Systems for the Production of Retroviral Vectors: A Safety Concern.

The development of efficient and safe production procedures are an essential goal conditioning viral vectors usefulness. For retroviral vectors production, two groups have suggested that Semliki forest virus (SFV) derived systems might generate interestingly elevated yields. The system relies on the transfection of three complementary SFV replicons. It is noteworthy that Muriaux and collaborator have shown that RNA is an important component for retroviral particles and that some SFV RNA could be packaged into retroviral particles. Furthermore, Lebedeva et al. and Rolls et al. have shown the arising of autonomous replication replicon from SFV vectors expressing various viral glyco-proteins envelopes. Thus, these data suggested that retroviral vectors might be vehicles for all sorts of SFV derived replicons.We designed a study aimed at thoroughly examining the packaging of full length SFV replicon into retroviral vectors. For this purpose we constructed two GFP expressing SFV replicons. One contained a prototypic Ψ sequence from MLV, while the other was missing this sequence. As SFV replicons normally give rise to genomic as well as subgenomic RNAs, we also modified their structure to prevent the formation of the later. This allowed us to set simple mobilization assays with straightforward read out based on the counting of GFP positive cells. In a first series of experiments we separately followed the mobilization of the modified SFV-GFP vectors in three conditions: using standard retroviral producing cells (PhoenixA cells); using the Li and Garoff Semliki based retroviral producing system and, lastly, in a tri-transfection lentiviral vector production. We observed mobilization events with all three conditions, albeit at different efficiencies. Using a secondary mobilization assay, we next showed that, in the Li and Garoff system, the MLV particles not only contained the GFP Semliki vector but also the Gagpol and the Env full length SFV replicons. Furthermore, a western blot analysis suggested that the retroviral particles might also contained uncleaved replicase.Finally we have shown that, in producing cells, both the retroviral proteins and the Semliki replicase were colocalised.All together, our data clearly demonstrate that retroviral particles have the ability to package genomic and subgenomic SFV RNAs, containing or not a retroviral Ψ. Therefore, these observations strongly advocate against the use of the Semliki system for clinical grade retroviral or lentiviral vector production.Molecular Therapy (2006) 13, S183–S183; doi: 10.1016/j.ymthe.2006.08.542 [ABSTRACT FROM AUTHOR]