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

Area of Science:

• Gene therapy vector development
• Molecular virology
• Biotechnology safety

Background:

• Third-generation retroviral and lentiviral vectors (RV/LVs) offer improved safety profiles.
• Updated regulatory guidance exists for replication-competent retrovirus (RCR) and replication-competent lentivirus (RCL) assays.
• A reassessment of RCR/RCL formation risk in RV/LV systems is needed.

Purpose of the Study:

• To review the historical context of RCR formation and safety features in RV/LV systems.
• To model the risk of RCL formation in four-component LV systems using established recombination rates.
• To propose a roadmap for reduced RCR/RCL testing based on current vector system safety.

Main Methods:

• Review of historical RCR generation and third-generation RV/LV safety features.
• Development of a minimal-path model for RCL formation in four-component LV systems.
• Analysis of recombination rates between plasmid DNA and retroviral RNA.

Main Results:

• The probability of generating a single RCL genome molecule at a 200-L scale is less than 1 in 10,000 per bioreactor.
• Using packaging cell lines with integrated vector components reduces RCL formation probability by 100-fold.
• Contemporary RV/LV systems demonstrate a low probability of RCR/RCL generation.

Conclusions:

• The low improbability of RCR/RCL generation in current RV/LV systems supports a move towards reduced testing.
• Consideration of a roadmap for reduced testing and assay development unification is recommended.
• Further contemplation on aspects of a reduced testing roadmap is encouraged for the field.