Chinese hamster ovary cell line engineering strategies for modular production of custom extracellular vesicles.

Continuously secreted by all cell types, extracellular vesicles (EVs) are small membrane-bound structures which shuttle bioactive cargo between cells across their external environment. Their central role as natural molecular messengers and ability to cross biological barriers has garnered significant attention in the use of EVs as therapeutic delivery vehicles. Still, harnessing the potential of EVs is faced with many obstacles. A cell line engineering approach can be used to exploit EVs to encapsulate a bespoke cargo of interest. However, full details regarding native EV-loading mechanisms remain under debate, making this a challenge. While Chinese hamster ovary (CHO) cells are well known to be the preferred host for recombinant therapeutic protein production, their application as an EV producer cell host has been largely overlooked. In this study, we engineered CHO DG44 cells to produce custom EVs with bespoke cargo. To this end, genetic constructs employing split green fluorescent protein technology were designed for tagging both CD81 and protein cargoes to enable EV loading via self-assembling activity. To demonstrate this, NanoLuc and mCherry were used as model reporter cargoes to validate engineered loading into EVs. Experimental findings indicated that our custom EV approach produced vesicles with up to 15-fold greater cargo compared with commonly used passive loading strategies. When applied to recipient cells, we observed a dose-dependent increase in cargo activity, suggesting successful delivery of engineered cargo via our custom CHO EVs.

GFP-tagging of extracellular vesicles for rapid process development.

Extracellular vesicles (EVs) act as nano-scale molecular messengers owing to their capacity to shuttle functional macromolecular cargo between cells. This intrinsic ability to deliver bioactive cargo has sparked great interest in the use of EVs as novel therapeutic delivery vehicles; investments totaling over $2 billion in 2020 alone were reported for therapeutic EVs. One of the bottlenecks facing the production of EVs is the lack of rapid and high throughput analytics to aid process development. Here CHO cells have been designed and engineered to express GFP-tagged EVs via fusion to CD81. Moreover, this study highlights the importance of parent cell characterization to ensure lack of non-fused GFP for the effective use of this quantitative approach. The fluorescent nature of resulting vesicles allowed for rapid quantification of concentration and yield across the EV purification process. In this manner, the degree of product loss was deduced by mass balance analysis of ultrafiltration processing, reconciled up to 97% of initial feed mass. The use of GFP-tagging allowed for straightforward monitoring of vesicle elution from chromatography separations and detection via western blotting. Collectively, this work illustrates the utility of GFP-tagged EVs as a quantitative and accessible tool for accelerated process development.