Understanding extracellular vesicle physiology for the development of bioinspired nanotechnology platforms
Lead Research Organisation:
University of Oxford
Department Name: Interdisciplinary Bioscience DTP
Abstract
Intercellular communication is essential for normal physiology of all multicellular organisms, for example in development, immune surveillance, neuronal plasticity and many other processes. An important part of this communication is mediated by extracellular vesicles (EVs) - nanovesicles which are secreted by eukaryotic cells and act by delivering their protein and RNA cargo to target cells/tissues. Better understanding of the physiology of EV protein and RNA bioactive transfer mechanisms between specific tissues/cells will improve our knowledge about intricate cell and tissue communication networks on a whole organism level. Furthermore, detailed understanding of the these information/cargo transfer networks will enable the development of bioinspired nanotechnology platforms for manipulating cell- and tissue physiology for probing novel biological principles on an organism level. Even more, the latter will also open up new therapeutic intervention avenues for currently non-druggable disease by developing targeted delivery systems for biopharmaceuticals.
Our goal is to advance the EV physiology field by combining aspects of systems biology and traditional reductionism approaches. We aim to define rate limiting steps in EV RNA cargo transfer, such as ligands, co-factors and antigens that modulate the differential circulation half-life and clearance of native and exogenous EVs, as well the half-life of different RNA species within EVs. Furthermore, we aim to define which EV RNA cargo sequences are targeted and taken up by which tissues, cell types and subcellular organelles, and which are the associated mechanisms. We will undertake this challenging cutting-edge research which will greatly advance current state-of-art of EV physiology field in novel but highly important aspects.
Collaboration with the non-academic partner will help to advance the project in significant ways, importantly by providing significant translational relevance for the discovered new aspects and detailed nuances of EV biology, as well as by providing supplementary training opportunities for the applicant for no less than 12 weeks during the study programme.
Our goal is to advance the EV physiology field by combining aspects of systems biology and traditional reductionism approaches. We aim to define rate limiting steps in EV RNA cargo transfer, such as ligands, co-factors and antigens that modulate the differential circulation half-life and clearance of native and exogenous EVs, as well the half-life of different RNA species within EVs. Furthermore, we aim to define which EV RNA cargo sequences are targeted and taken up by which tissues, cell types and subcellular organelles, and which are the associated mechanisms. We will undertake this challenging cutting-edge research which will greatly advance current state-of-art of EV physiology field in novel but highly important aspects.
Collaboration with the non-academic partner will help to advance the project in significant ways, importantly by providing significant translational relevance for the discovered new aspects and detailed nuances of EV biology, as well as by providing supplementary training opportunities for the applicant for no less than 12 weeks during the study programme.
Organisations
People |
ORCID iD |
| Matthew Wood (Primary Supervisor) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M011224/1 | 01/10/2015 | 31/03/2024 | |||
| 2104930 | Studentship | BB/M011224/1 | 01/10/2018 | 30/09/2022 |
| Description | The general idea of my project's first aim was to develop new tools and methods to enhance and advance the extracellular vesicle (EV) biotherpeutic platform. Originally we intended to develop EV DNA barcoding technology. This technology was intended to serve as a more specific and sensitive method of quantifying the number of EVs that reach different tissues and organs within recipients. However, during the development of this method we noted inconsistent and inefficient bonding efficiencies. Additionally, we gathered some data that suggested that only certain proportion of EV populations were bonded with global labeling mechanisms. This suggested that only certain subpopulations of EVs may be able to be bonded with certain global labels, and a cocktail of labels may be necessary to label an entire EV population. We submitted applications with collaborators to attempt to investigate this further. Furthermore, while qPCR is a very sensitive technique, numerous issues were experienced with the contamination of samples or standards in the development of the barcoding protocol, as well as issues in quantifying the number of barcodes in each samples when contamination was not present. While we did not fully satisfy the original first aim of the project to develop and optimise DNA barcoding, an extensive effort was conducted to optimse and assess the capabiliteis of the toolset. However, due to issues surrounding EV purification that arose during the project, we incorporated the development and validation of Capto Core purification improve EV purification from high density cultures into the first aim. This would enhance the EV biotherpeutic platform as to obtain high, clinically relevant EV concentrations necessary for treating human patients, EV production must be upscaled into high density cell cultures that produce an equally high density of EVs and other impurities are needed. We developed and validated the use of novel liquid chromatography based purification method for the purification of extracellular vesicles. We validated the use of this method against size exclusion chromatography (SEC), one of the methods considered to be among the best for EV purification. Our results showed that Capto Core chromatography was a far superior method to SEC for the purification of EVs from high density cultures, with high EV yields and protein:particle ratios, low EV losses and low host cell protein. When combined with further processing by anion exchange low nucleic acid content was also seen. This technique was employed during later stages of the proejct. The other aims of my project were associated with understanding and harnessing EV heterogeneity for the betterment of the EV biotherapeutic platform. Firstly the effects and proteomes of SKOV3 ovarian cancer EVs from different size based subpopulations was explored. It was discovered that EVs of different sizes have differing tendencies to influence cell adhesion in vitro. What's more, proteomics analysis revealed significantly different proteomes of EVs in size based subpopulations which could further indicate different functions. Furthermore, the effects of cellular phenotypic drift on the vesicular proteom were explored by taking who subclones of MDA-MB-231 breast cancer cells, one that metastasized primarility to the brain, and hte other that primarily metastasized to the lungs and grow three seperate cultures of each of these cell lines. By testing EVs serived from each of them for the expression of typical EV markers, the expression of highly expressed proteins seemed to remain fairly stable, however lower expressed protein fluctuated widely, which could furhter suggest that cellular phenotypic drift, epigenetic factors or geneomic instability to could affect EV proteomes and therefore function. Finally, EVs derived from single cell sorted populations of MSCs were tested in numerous functional assays to identify whether the source of EV heterogeneity is cell derived or stochastic, and to see whether a functionally distinct subpopulations of EVs with a correlating marker can be identified in order to inform industry to isolate these EVs as opposed to entire EV populations to improve therapeutic efficacy. In vitro cell migration assays revealed differences in cells migration with cell adhesion and DPP4 assays yet to be completed. Lactate Glo assays were performed to assess in vitro T cell activation, however no differences in function were observed. Despite these there were significant differences in lactate concentrations within EVs which was notable and could serve as an indicator of differences in the metabolism of the parent cells. Proteomics analysis revealed notable differences in the EV proteomes of each EV subclones with varying levels of functional markers of cell adhesion and wound healing. Differences in cell and EV phenotypes are to be established in the coming months, along with correlation analysis between EV functional assays and proteomics analysis. |
| Exploitation Route | Researchers may adopt the use of multiple global labeling chemistries to ensure the entirety of an extracellular vesicle (EV) population is labelled. Once published researchers may opt to use novel Capto Core bind-elute liquid chromatography for the purification of their EVs whether from high cell density cultures or conventional culture, with or without anion exchange post processing to remove further DNA and RNA. Researchers may also consider the impact that cellular phenotypic drift and EV size can have on the function of their EV or potential EV therapeutics. What's more, researchers may attempt to identify EVs that express certain markers on their surface to improve their therapeutics efficacy, as it has been shown that EVs isolated from single cell sorted cultures of MSC have different functional properties, despite being derived from the same parent cell. |
| Sectors | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | Finding Genius Podcasts |
| Form Of Engagement Activity | A broadcast e.g. TV/radio/film/podcast (other than news/press) |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Media (as a channel to the public) |
| Results and Impact | Recorded two separate podcasts for the Finding Genius Podcast (previous known as Future Tech), hosted by Richard Jacobs. The first podcast was titled On the Function and Therapeutic Potential of Extracellular Vesicles and was released on the 3rd of February 2020 on podcasting platforms and YouTube. The second podcast was titled Understanding Extracellular Vsicles and their Effect on Cancer Development Discussed with Scott Bonner and was released on 11th June 2021. During the podcast I discussed how research on vesicle heterogeneity might elucidate how differences between vesicle phenotypes result in differences and potentially therapeutic functionality, How man EVs one cell can produced and why it's challenging but becoming increasingly feasible to example singular vesicle phenotypes, what role EVs play in cell communication, how EVs are isolated, and the function of EVs within the body. |
| Year(s) Of Engagement Activity | 2020,2021 |
| URL | https://podcasts.google.com/feed/aHR0cHM6Ly9maW5kaW5nZ2VuaXVzcG9kY2FzdC5saWJzeW4uY29tL3Jzcw/episode/... |
| Description | International Society for Extracellular Vesicles poster presenations 2020 and 2021 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Presented two separate posters at the annual international society of extracellular vesicles conference (one poster in 2020 and another in 2021). The purpose was to publicise ongoing research. Both events were attended by over 500 people and were hosted online due to the COVID-19 pandemic. In each year both my posters gained over 250 views and generated numerous questions from attendees. My 2020 poster was titled "Extracellular vesicles heterogeneity and the impact of cellular phenotypic drift on the vesicular proteome" This poster concluded that temporal changes in the EV proteome are inconsistent among independent cultures of the same cells, highlighting the effects of cellular phenotypic drift, epigenetic influences or cellular genomic instability of the vesicular proteome, frequent changes in the vesicular proteome may impact EV toxicity or therapeutic efficacy, and protein expression varied erratically among proteins that were more scarcely expressed and abundantly expressed proteins were more stably expressed. My 2021 poster was titled "High purity extracellular vesicles isolated from high-density cultures using novel Capto Core bind-elute chromatography". The poster concluded that Capto Core chromatography resin is composed of porous beads with a ligand activated core that facilitate the binding of impurities such as proteins with the core and elution of the purified EV product, Capto Core bind-elute chromatography is superior to SEC for the purification of EVs from proteins and nucleic acid contaminants, presenting roughly two-fold higher particle:protein and particle:DNA+RNA ratios compared to equivalent complete purification by SEC and Western blot and TEM data show preserved EV phenotypes, and intact, non-aggregated particles compared to SEC, and Capto Q presents a promising addition to the purification protocol with high particle:DNA+RNA ratios and a 95% reduction in total DNA+RNA. |
| Year(s) Of Engagement Activity | 2020,2021 |
| Description | Tartu Extracellular Vesicles Symposium |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Was invited to present at the 2020 Tartu Extracellular Vesicles Symposium . The symposium was hosted online due to the COVID-19 pandemic. The purpose of the talk was to publicise the work I had been conducting in the extracellular vesicle heterogeneity space. The presentation discussed the impact of cellular phenotypic drift of MDA-MB-231 cells on extracellular vesicle heterogeneity. |
| Year(s) Of Engagement Activity | 2020 |