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.

Grant Number: 1R21EB036789-01A1
NIH Institute/Center: NIH
Principal Investigator: Mark Blenner