Carrillo Sanchez, Braulio;
(2022)
Design and production of engineered extracellular vesicles in Chinese hamster ovary cells.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
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.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Design and production of engineered extracellular vesicles in Chinese hamster ovary cells |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering UCL > Provost and Vice Provost Offices > UCL BEAMS UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10154885 |
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