(235c) Engineering Cell Lines for the Production of Highly Replication Defective Herpes Simplex Virus Type 1 Vectors

In order to mass produce clinical grade highly replication defective HSV-1 vectors, efficient cell lines must be generated to complement missing viral functions while avoiding recombinant virus generation. The goal of this work is to increase the yield of replication defective virus per cell by engineering a cell line that would provide efficient timing and adequate expression levels of the immediate early gene ICP4. Conventional replication defective HSV-1 vectors have deletions in immediate early genes ICP4 and ICP27 and are propagated on complementing cell lines that stably express these genes in trans. Here a joint deleted ICP4, ICP22, and ICP27 mutant virus (JDTOZEH1) was used to examine the robustness of engineered cell lines during a low MOI (0.01) production. The ICP0 promoter was chosen to drive ICP4 complementation due to a higher concentration of viral activation elements (TAATGARAT sequences). This strategy lead to higher induction levels and faster kinetics of ICP4 expression which resulted in 10 fold increases in viral yield per cell. Quantitative PCR for ICP4 transcription revealed a 10-fold increase and faster kinetics of ICP4 RNA transcription suggesting this promoter is sufficient for ICP4 complementation. These results were verified by Western blotting for ICP4 showing both earlier and higher levels of expression. Additionally, stocks produced from this cell line contained no replication competent viruses since the fragments used to transfect the complementing cell lines have limited homology with the deletions of the viral genome. These cell lines will serve as building blocks for large scale production of consequently less toxic versions of therapeutic HSV-1 viral vectors.