Researcher(s)
- Olivia Quick, Chemical Engineering, University of Delaware
- Susan Kuhn, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware
Abstract
Gene therapy presents substantial potential for addressing numerous genetic disorders. Recombinant adeno-associated virus (rAAV) is an effective vector for gene therapy by delivering packaged therapeutic genes to both dividing and non dividing cells. While rAAV is a successful delivery vehicle for gene therapies, its production in host cells is unpredictable due to various factors. One of the suspected causes of low rAAV production is due to the accumulation of the Rep protein within the vector. Rep 78/68 is necessary for rAAV production as it enables DNA replication, however it has been proven to be cytotoxic. This study investigates the effects of attaching an oscillating degron tag to the Rep 78/68 protein on rAAV production by degrading and accumulating the rep gene at various points in the cell cycle.
Degron tags are a type of protein tag that can facilitate the degradation of the tagged protein. We cloned a degron tag onto the Rep protein of the vector using gibson assembly. We tested two degron tags to evaluate which would be more effective. Geminin accumulates during the S phase of the cell cycle and disappears at the time of the anaphase to metaphase transition during mitosis whereas Cdt1 accumulates during the G1 phase of the cell cycle and degrades during the S phase. We will arrest the cells in certain phases of the cell cycle to measure the amount of Rep present at certain time points. By testing two degron tags that degrade during opposite times in cell replication, we will evaluate the degron tags ability to regulate Rep gene expression in a way that allows for improved rAAV production and ultimately see which one performs the best. The objective of this research is to discover another way to improve rAAV production in host cells.