Multidose transient transfection of human embryonic kidney 293 cells modulates recombinant adeno‐associated virus2/5 Rep protein expression and influences the enrichment fraction of filled capsids

Recombinant adeno-associated virus (rAAV) is a commonly used in vivo gene therapy vector because of its nonpathogenicity, long-term transgene expression, broad tropism, and ability to transduce both dividing and nondividing cells. However, rAAV vector production via transient transfection of mammalian cells typically yields a low fraction of filled-to-total capsids (~1%-30% of total capsids produced). Analysis of our previously developed mechanistic model for rAAV2/5 production attributed these low fill fractions to a poorly coordinated timeline between capsid synthesis and viral DNA replication and the repression of later phase capsid formation by Rep proteins. Here, we extend the model by quantifying the expression dynamics of total Rep proteins and their influence on the key steps of rAAV2/5 production using a multiple dosing transfection of human embryonic kidney 293 (HEK293) cells. …