Addressing replication-competent lentivirus (RCL) is critical in LVV production. We have mitigated RCL through the design of our system and also have confirmed through RCL testing.

Design

Several key factors which influence the frequency of recombination events were taken into consideration in the design of our LV Edge system:

1. Reduction of homology between vector and helper sequences.

2. Reduction in homology between vector/helper sequences and cellular DNA.

3. Division of helper sequences to more than one expression cassette to increase the number of recombination events required to generate RCL.

4. Integration of viral elements have been distributed throughout the genome and not a single loci, hence reducing likelihood of RCL.

RCL evaluation

We have tested RCL for the three clinically relevant molecules generated by our packaging cell line and samples have been cleared according to vendor qPCR based RCL detection assay to meet FDA requirements. (Briefly, our viral supernatant was used to transduce cells at 5 MOI.  Transduced cells were passaged, and then RNA was extracted from the supernatant of these growing transduced cells and screened for the presence of virus.  If any recombination had occurred during the transduction which allowed replication of virus, it would have been detected.)