Influencing Cell Cycle Arrest Activity of Cytotoxic Rep Using Oscillating Degron Tags for rAAV Production

• Nicole Webb, Chemical Engineering, University of Delaware

• Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware

Recombinant Adeno-Associated virus (rAAV) is a powerful tool to deliver gene therapy into the cells of the body. However, Rep 78/68 is a cytotoxic protein used in the production process for virus replication. Rep arrests cells in the S-phase, which prevents the cell from completing DNA replication and therefore lowers rAAV production capabilities. We can use natively oscillating proteins in the cell, CDT1 and Geminin, to reduce Rep expression. Truncated versions of these proteins can act as degron tags that signal the cell at a specific phase of the cell cycle to degrade the protein. 

Previous experiments used flow cytometry to visualize the cell cycle of a cell transfected with a degron-tagged Rep plasmid. However, it was difficult to visualize any improvements caused by the degron tags due to the constitutive CMV promoter overexpressing Rep. Therefore, we created a new version of the plasmid with an inducible Tet promoter. By inducing the promoter with small doxycycline concentrations, Rep expression would be weakened, therefore allowing us to visualize if the degron tags were able to competitively degrade the lower rate of Rep expression. 

We transfected this plasmid into HEK293 cells, then harvested the cells and prepared them for flow cytometry through a click chemistry reaction that fluorescently labelled EdU. This allows us to visualize the full cell cycle profile as the cells uptake EdU in a normal S-phase progression, or if they remain arrested in the S-phase.

Our results showed the same trends as those from the constitutive CMV promoter: CDT1 did not improve cell cycle arrest, while Geminin showed slight improvement. Therefore, we found that while we were able to influence the percentage of arrested cells by incorporating degron tags into the Rep plasmid, an inducible promoter did not yield clearer results than a constitutive promoter.