(49e) Light-Inducible Regulation of Autophagy in Chinese Hamster Ovary Cells for Production of Therapeutic Proteins

Autophagy is a potential tool to significantly increase the production of therapeutic proteins. As a major pathway in the cellular energy balance, autophagy is an evolutionarily conserved process by which proteins, organelles, and lipid matter are recycled into amino acids and free fatty acids to synthesize new cellular components. Several groups have demonstrated that modulating autophagy significantly influences therapeutic protein production^1,2. However, precise regulation of autophagy for higher titers remains challenging. Current methods of modulating autophagy lack temporal control or specificity to the autophagy pathway, and the way in which autophagy controls protein synthesis is not well-understood. Thus, developing a method for sensitive control over the process is critical for elucidating the relation between autophagy and protein production and effectively regulating autophagy for higher titers. We are constructing light-inducible systems for transcriptional and direct regulation of autophagy in Chinese Hamster Ovary (CHO) cells, which are the major mammalian cell line for industrial therapeutic protein production.

The transcriptionally regulated system enables light-inducible gene expression by using Clustered Regularly Interspaced Short Palindromic Repeats-dCas9 (CRISPR-dCas9), a modular system that can be used to regulate any gene of interest³. We have demonstrated the tunability of the system using green fluorescent protein (GFP), achieving up to five-fold higher GFP expression for activated cells (Figure 1). We are currently using the system to express constitutively active mutants of AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin 1 (mTORC1), which are well known inducers and inhibitors of autophagy, respectively.

We have also designed a light-inducible system which specifically regulates subcellular mTORC1 localization. Localization of mTORC1 to the lysosome has been shown to be sufficient for its activation⁴, after which it inhibits autophagy. Thus, photoregulation of mTORC1 localization to the lysosome can be used to control autophagy. Compared to transcriptional regulation, direct regulation of protein activity has the advantage of eliminating lag-times of the system due to the time taken to express the protein when turning the system on and the half-life of the protein when turning the system off.

Using the two light-inducible systems, we are investigating the effects of AMPK and mTORC1 activity on autophagy rate and protein synthesis. The specificity of the systems allow precise control over autophagy, which will facilitate maximizing titers and advance knowledge on how autophagy regulates protein production.

References

1. Nasseri, S. S. et al. Increased CHO cell fed-batch monoclonal antibody production using the autophagy inhibitor 3-MA or gradually increasing osmolality. Biochem. Eng. J. (2014) doi:10.1016/j.bej.2014.06.027.
2. Lee, J. S. & Lee, G. M. Rapamycin treatment inhibits CHO cell death in a serum-free suspension culture by autophagy induction. Biotechnol. Bioeng. 109, 3093–3102 (2012).
3. Polstein, L. R. & Gersbach, C. A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015).
4. Sancak, Y. et al. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290–303 (2010).