Risks of replication-competent retro/lentivirus from associated vector systems: Is it time for a roadmap toward...

The development of retroviral and lentiviral vectors (RV/LVs) has culminated in third-generation vector systems with excellent safety profiles. However, there has been no significant (re)assessment of RCR/RCL formation risk within RV/LV systems regarding the underlying molecular recombination events, despite updated guidance for RCR/RCL assay development/testing issued by the key regulatory agencies. This review reflects on genuine RCRs generated in early vector systems that drove the development of the common safety features of all third-generation systems. We then present a "minimal-path" model for RCL formation within four-component LV systems, which draws on published recombination rates between plasmid DNA in cell lines and retrovirus genomic RNA during reverse transcription. This model indicates that the probability of generating a single RCL genome molecule at 200-L scale is ≤ 1 in 10,000 per bioreactor and is reduced by 100-fold if packaging cell lines containing integrated vector components are used. We propose that the improbability of generating RCR/RCL within contemporary vector systems justifies consideration of a roadmap to reduced testing and provide potential aspects of such a roadmap for the field to contemplate. We also review past and current guidelines for RCL testing and assay development, recommending key aspects of method development toward unification in the field.