Establishing a stable production cell line for recombinant AAV through synthetic dynamic regulation and reducing regulatory and metabolic limitations - PROJECT SUMMARY The current manufacturing methods for recombinant Adenovirus-Associated Virus (rAAV) is transient transfection, which faces numerous challenges, such as low productivity of rAAV from host cells, difficult scalability of the rAAV-producing bioprocess, and high levels of impurities (e.g. empty/partial capsid) materialized during production. Furthermore, nucleic acid production represents the majority of the rAAV manufacturing costs. A stable producer cell line could address all of these concerns; however, it requires the integration of not only the gene of interest (GOI), Rep, and Cap genes for genome replication and encapsidation, and the helper proteins that initiate the rAAV replication. The cytotoxicity induced by the continuous expression of rep and helper genes after integration have hindered efforts to establish a stable cell line for rAAV. This project brings together several innovations necessary to achieve this long-desired goal in the field of viral vector biomanufacturing. Efforts to move from transient transfection manufacturing processes have been hindered by the instability of producer and packaging cells lines caused by the cytotoxic effects of the Rep78 expression and its regulation of the E1a, E2a, and E4 adenovirus early promoters. Precise control over gene expression is necessary to overcome this limitation. Therefore, our project builds our refactored the rAAV expression pathway enabling inducible control of the expression of rAAV genes and helper genes. This level of control also enables dynamic regulation and tuning the expression levels to achieve high quality rAAV with a high filled capsid ratio. The use of oscillating degron tags will enable periodic reduction of Rep78 levels as Rep78 arrests the cell cycle. Proper stoichiometry and expression dynamics will be achieved through the design of post-transcriptional control of gene expression by a series of nested gene circuits that autonomically control the timing of gene expression. The general expression patterns and dynamics will be guided by mechanistic modeling of rAAV biogenesis in stable cell lines, while the precise tuning of the system will be more empirical. These efforts are combined with cell line engineering strategies informed by transcriptomic data of rAAV producing HEK293 cells, which targets ER stress and protein processing genes, innate immune response, and energy metabolism. If successful, this project would establish stable cell line production of rAAV, significantly driving down manufacturing costs, and increasing gene therapy accessibility.
