Cai, Wen2024-01-052024-01-052023-09https://hdl.handle.net/11299/259525University of Minnesota M.S. thesis. September 2023. Major: Microbial Engineering. Advisor: Wei-Shou Hu. 1 computer file (PDF); iii, 51 pages.Recombinant adeno-associated virus (rAAV) is one of the most promising gene delivery vectors for somatic gene therapy. Currently, its prevailing manufacturing technologies are relying on transiently transfecting host cells with three plasmids or infection of producer cell lines with helper viruses. Both methods pose real issues in process development, such as difficulty to scale-up or cleaning up helper virus from final products. Commonly used host cell lines for rAAV manufacturing are HEK293, and Sf9. Our lab has previously designed a helper virus-free, plasmid-free, stable cell line production system for rAAV2 via synthetic biology approach. The stable cell line was constructed by integrating multiple copies of rAAV2 genome, AAV2 Cap, AAV2 Rep and Ad5 helper genes which are under inducible promoter control and organized as three separate segments in Genome module, Replication module and Packaging module, into HEK293 cells genome. The stable cell line produced infectious rAAV2 particles upon induction. In this study, we aimed to explore the possibility of using Chinese hamster ovary (CHO) cell as the host cell and creating a stable producer cell line for rAAV2 production. Compared to HEK293 and Sf9, CHO cell holds many advantages. As the most commonly used industrial cell line for therapeutic protein production, it could reach high density in suspension cell culture using serum-free media and is resilient and robust in manufacturing conditions. This study showed that CHO cells were capable of translating AAV2 viral proteins, replicating rAAV genomes, and packaging them into rAAV vector in transient transfection using the Genome, Replication and Packaging modules. Expression of two Ad5 gene, E1A and E1B, could further enhance rAAV titer. E1A and E1B could be stably integrated into the CHO-K1 host cell genome along with the three modules under inducible promoter control. Current producer cell lines had a low productivity and their productivity appeared unstable. Nevertheless, our study demonstrated the potential of CHO cell lines as a novel production platform for rAAV manufacturing.enAAVCell Line EngineeringGene TherapyThesis or Dissertation