Lee, Zion2021-10-132021-10-132021-08https://hdl.handle.net/11299/225020University of Minnesota Ph.D. dissertation. 2021. Major: Chemical Engineering. Advisor: Wei-Shou Hu. 1 computer file (PDF); ix, 74 pages.The recombinant adeno-associated virus (rAAV) gene therapy field has experienced landmark regulatory approvals in recent years by demonstrating high efficacy in treating monogenic diseases. There is therefore an immediate and increasing need for highly productive manufacturing platforms to generate large quantities of rAAV vectors. Current rAAV synthesis methods require multiple-component plasmid transfections or viral infections, which have increasing costs, variability, and technical challenges at large scales. To address the growing need for robust methods to produce and characterize rAAV vectors, we used synthetic biology tools to gain control over viral gene expression dynamics. First, we engineered a rAAV infectious titer assay cell which contains the AAV and helper genes necessary to replicate transduced genomes. Once exposed to small molecule inducers and a rAAV vector, the assay cell line performed similarly to the standard adenovirus (Ad) coinfection method in determining the infectious titer of the vector preparation. Omitting the use of Ad greatly simplifies the potency assay and reduces the variability of input reagents. Building on that success, we further engineered cells that not only have replicative capacity, but also packaging capacity and a latent copy of a rAAV genome. Small molecule induction allowed expression of the necessary viral genes to synthesize infectious rAAV vectors. The variation of inducer ratios enabled manipulation of vector quality, in terms of the fraction of DNA-containing particles. These synthetic cellular technologies address the needs of a reliable potency bioassay and a scalable and robust production platform for rAAV manufacturing.enadeno-associated virussynthetic biologyThesis or Dissertation