Delivering on the potential of RNA therapeutics Microfluidic mixing for manufacturing hybrid nanoparticles. Synthetic lipids are dissolved in the organic phase prior to mixing with the aqueous phase containing extracellular vesicles. Upon mixing under controlled conditions, hybrid nanoparticles can be formed.
RNA therapeutics hold great potential as a means of treating a range of diseases, yet they are also immunogenic and it is difficult to transport these molecules to specific locations in the body. We spoke to Dr Pieter Vader, Diego Aguilar Rodriguez and Willemijn de Voogt about the work of the OBSERVE project in developing extracellular vesicle-based systems to deliver RNA drugs into the body. The potential of
RNA therapeutics is enormous, with researchers investigating possible applications in vaccine development, cancer treatment and several other areas of medicine, yet RNAs are also difficult molecules to work with. These molecules are fragile and can be degraded by various different enzymes, while there are also further issues to overcome before they can be applied more widely. “They are immunogenic, while they are also large and negatively charged, so they cannot spontaneously enter cells, which is necessary for them to perform their function,” says Dr Pieter Vader, Associate Professor in CDL Research and the Department of Experimental Cardiology at the University Medical Center Utrecht. As the Principal Investigator of the OBSERVE project, in which he is working together with PhD students Diego Aguilar Rodriguez and Willemijn de Voogt, Dr Vader is investigating the possibility of using extracellular vesicles (EVs) as a way to deliver RNA therapeutics and help overcome the issues which are currently limiting their application. “These
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EVs are vesicles that are secreted by the cell, towards the extracellular milieu,” he outlines.
Extracellular communication These EVs are thought to have a variety of roles, with Dr Vader primarily interested in their role in intercellular communication. Some EVs are able to deliver content including proteins, RNAs and other small molecules in a
it?” he explains. A tool based on the CRISPRCas9 machinery is being used in the project to study EV-mediated RNA delivery on the single cell level. “With this tool we can see which cells in a dish or an organoid have taken up RNA via our vesicles,” continues Dr Vader. “We can then try comparing different types of vesicles in terms of which deliver most efficiently. We can perhaps then modify them
We’re studying naturally occurring extracellular vesicles – we study what they carry, how they carry it, as well as how they deliver it. We’re really trying to map that – which vesicles can deliver RNA and how do they do it? functional manner to other cells, a topic that Dr Vader and his colleagues in the project are investigating. “We’re studying naturally occurring EVs – we study what they carry, how they carry it, as well as how they deliver it. We’re really trying to map that – which vesicles can deliver RNA and how do they do
a little bit and ask the question; does their delivery capacity improve?” This is an important issue in terms of using these vesicles therapeutically, an issue high on the project’s agenda. Researchers are using naturally-occurring vesicles, but certain changes are necessary in order to
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load them with exogenous, therapeutic RNA. “We need to modify the EVs in order to give them the ability to carry RNA, so we are applying certain tricks, including hybridization with synthetic nanoparticles and other approaches, to load sufficient amounts of exogenous RNA into them,” says Dr Vader. This is a significant challenge, as typically there is nothing inside the vesicle to ‘hold’ on to the RNA; Dr Vader is developing hybrid nanoparticles, comprised of an EV and a liposome, to address this. “The liposomes carry a bit of positive charge and the RNA is negatively charged, so we were able to get one of the lipid components to hold on to the RNA,” he explains. “We then essentially merged the two nanoparticles – the liposomes and the EVs – together through extrusion.” The liposome is a bi-layer structure, effectively a very simple model system of an EV. With the addition of a bit of charge to hold onto the RNA, these two nanoparticles are merged together through extrusion, resulting in a hybrid structure. “This structure resembles an EV, but we’ve added some exogenous lipids, and it can now carry this therapeutic RNA,” outlines Dr Vader. By modifying the amounts of liposome and EVs used in forming these structures, Dr Vader and his colleagues are able to change the way they function. “We see that if we use a higher ratio of EVs compared to liposomes, then the mechanism of uptake is dictated by the EVs. Whereas if we have a lower ratio of EVs, we see that the liposome dictates where they go,” he says. “The problem is that at the moment we can’t really increase the proportion of EVs in the hybrid nanoparticle any further, just from a practical standpoint. We want to go even higher and investigate what happens, but first we have to optimise our manufacturing process.”
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Microfluidic mixing This is an issue that is being addressed in the project, with researchers investigating other methods of producing these hybrid nanoparticles. One possibility that Dr Vader is looking at is microfluidic mixing, which involves performing rapid mixing in a highly controlled manner, using very small volumes. “This is done on a chip. You have an inlet of the vesicle components, an inlet of the synthetic lipids, and then a highly controlled, rapid, chaotic mixing, before we get our end product,” he explains. The ultimate objective here is to develop a hybrid structure that can be loaded with a
Super-resolution microscopy image (dSTORM) of single extracellular vesicles (EVs). EVs are labeled with the general membrane dye Memglow560 (cyan). Transmembrane proteins CD81 (magenta) and CD63 (yellow) are detected on the EVs through staining with specific antibodies. Imaging was performed on the Nanoimager S (ONI). Top right: Zoom-in of an individual EV containing both CD81 and CD63.
therapeutic RNA; while this approach could be used to treat a variety of conditions, the focus in the OBSERVE project is on heart failure. “The structure will be based on stem- or progenitor cell-derived vesicles, which hold intrinsic regenerative properties. We will evaluate those in a mouse model of heart failure, of ischemia re-perfusion damage,” outlines Dr Vader. “We believe that we can combine the RNA delivery properties of EVs with their natural properties.”
A more effective way of delivering RNA therapeutics into the body would represent a significant step towards realising their potential. The main benefits of RNA therapeutics are firstly that they are highly specific, and secondly that they target a disease at its source. “They target the genetic disorder, or a gene that is wrongly expressed for example,” says Dr Vader. The EVs could also be used to deliver different types of RNA molecules, opening up wider possibilities. “The general demand from the delivery system is very similar for these RNA molecules, because they all have to be delivered to the cytosol of the target cells. So in principle if you can deliver one RNA, you can also deliver another,” continues Dr Vader. “However the loading, and the strategy for loading, may be different for a small RNA in comparison to a large one. Ideally you would want to load as much RNA as you can into a single EV.” These EVs also represent a safer and more versatile approach to delivering RNA than synthetic lipid nanoparticles (LNPs), which have been used in Covid-19 vaccines. While synthetic LNPs work effectively in delivering vaccines, they can lead to adverse reactions in other applications, which is not the case with EVs. “As a natural vehicle, EVs may not cause adverse reactions, so they are a much safer option,” explains Dr Vader. There is more work to do before EVs can be applied more widely however, and at this stage Dr Vader is still working to improve the hybrid nanoparticles. “We’re looking at the liposome-EV hybrids, using microfluidic mixing. We’re also looking at other hybrid structures, using for example polymeric nanoparticles,” he outlines. “Once we have prototypes that work, and that we can make at sufficient scale for a mouse model, we can look to move on. We are hoping to start some very preliminary testing in mouse models in the coming year .”
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OBSERVE Overcoming cellular barriers to therapeutic RNA delivery using extracellular vesicles Project Objectives
The OBSERVE project aims at exploring the potential of extracellular vesicles (EVs) as a means of delivering RNA therapeutics to cells. These EVs are secreted by cells into the extra-cellular milieu, and play an important role in inter-cellular communication. These EVs essentially transport different materials between cells, including proteins, RNAs, lipids and other small molecules. Researchers are probing how EVs deliver RNAs, and looking to explore the wider therapeutic possibilities.
Project Funding
This project has received funding from the European Union’s Horizon 2020 research and innovation programme, Starting Grant (StG), LS7, ERC-2019-STG.
Contact Details
Dr Pieter Vader, PhD Associate Professor CDL Research & Department of Experimental Cardiology Room G03.644 University Medical Center Utrecht Heidelberglaan 100 3584 CX Utrecht The Netherlands T: +31 88 7557654 E: pvader@umcutrecht.nl W: https://www.umcutrecht.nl/en/research/ researchers/vader-pieter-p
Dr Pieter Vader, PhD
OBSERVE Project Research Roles Researchers in the OBSERVE project are investigating several issues around EVs, with the aim of eventually using them to deliver RNA therapeutics. We spoke to Diego Aguilar Rodriguez and Willemijn de Voogt, PhD students at UMC Utrecht, about their role in the project, the methods they’re using, and the wider possibilities of RNA therapeutics. EU Researcher: Could I ask you both about your respective roles in the project? Willemijn de Voogt: My role is on the fundamental part of the project. I’m trying to unravel the biology behind EVs – how are they taken up? Which proteins or RNAs are involved in functional EV transfer? I’m looking at the pathways of intra-cellular trafficking, and the pathways underlying the transfer of RNA molecules. Diego Aguilar Rodriguez: I’m focused more on the potential therapeutic applications of EVs, with the goal of developing novel therapeutic tools to deliver mRNA. We’re looking to harness the properties of these EVs and trying to apply them in drug delivery. We’re trying to learn about these EVs and the potential benefits of their use in terms of things like uptake, targeting and preferential accumulation. EUR: Could you then look to bring these attributes to synthetic drug delivery systems? DAR: This is what we’re trying to do. Both EVs and synthetic drug delivery systems have limitations - we’re trying to get the best of both worlds.
Pieter Vader is Associate Professor at CDL Research and at the Department of Experimental Cardiology at the University Medical Center Utrecht. His main research interests are in the field of therapeutic applications of extracellular vesicles, including unraveling the mechanisms underlying extracellular vesicle-mediated cargo transfer. In 2021, Pieter was award the Prix Galien Research Award for his work on drug delivery.
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EUR: How does the relationship between basic and applied research work? Do you need to collaborate quite closely? WdV: Not yet, as the project is still at quite an early stage, but hopefully more so later on. For instance, if I find that certain proteins are essential for uptake or functional delivery in a certain cell type, then potentially Diego can design hybrids that contain specifically these proteins. EUR: How will you use microfluidic mixing to create hybrids? DAR: One of the phases is the organic phase, with synthetic lipids. In the aqueous phase we’ll have EVs with the therapeutic reagent, in this case mRNA. Then we will hybridise them with a microfluidic chip, then at the end we purify the sample. EUR: Do you see wider potential in RNA therapeutics? WdV: Very much so, we’ve seen the therapeutic potential of RNAs in recent times, for example with the development of vaccines against Covid-19. By exploiting these delivery vehicles, we can look to target specific diseases in multiple ways, such as by trying to express certain genes or knock down certain genes.
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