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

Design and production of engineered extracellular vesicles in Chinese hamster ovary cells

Extracellular vesicles (EVs) are lipid bilayer enclosed packages secreted by most mammalian cells. EVs are an important means of intercellular communication owing to their capacity to shuttle functional macromolecular cargo between cells. This functionality has sparked great interest in the use of EVs as novel therapeutics. Still, harnessing the potential of EVs is faced with many obstacles. The manufacture of EVs is challenged with non-standardised isolation methods and a lack of rapid analytics to aid process development. A cell engineering approach can be used to exploit EVs to encapsulate protein biocargo of interest. However, details regarding native EV loading mechanisms remain the subject of debate, making this a challenge. In this thesis, Chinese hamster ovary cells (CHO) were used to generate stable cells expressing genetic constructs designed for the encapsulation of cargo inside EVs. In the first half of the thesis, a process for recovery of EVs from high density CHO cell culture was established using ultrafiltration and size exclusion chromatography. To aid further process development, a fluorescence tagging approach to quantification was developed using engineered EVs. Cells were designed and engineered to express GFP-tagged EVs via fusion to CD81. The tetraspanin protein CD81, is a key EV marker and was chosen for fusion protein constructs to exploit its known abundance in EV populations. Resulting GFP-tagged EVs were then used for rapid quantification of yield across the EV isolation process and shown to reconcile up to 97% of initial feed from mass balance analysis. The focus in the second half of the thesis was to design and develop a modular cargo loading format to produce EVs with bespoke cargoes. To this end, genetic constructs employing split GFP technology were designed for tagging of both CD81 and protein cargoes to enable EV loading via self-assembling activity. To demonstrate this, NanoLuc luciferase and mCherry were used as model reporter cargoes to validate engineered loading into EVs. Experimental findings indicated that EVs contained up to 15-fold greater cargo using the genetic constructs developed in this thesis compared to commonly used passive loading strategies. Collectively, the findings presented in this thesis demonstrate the use of GFP tagging technologies to engineer EVs for different applications, spanning from process development to protein cargo loading