Engineering of Extracellular Vesicles for Oral Delivery of Nucleic Acid Therapies

Oral administration is the preferred way of taking medicines because it is convenient, painless, safe and the medicine can be self-administered by the patient. However, certain drugs, including nucleic acids, which are used as drugs to manipulate the production of important proteins by the body, currently cannot be administered orally. Nucleic acid-based drugs, an example of which is the Covid-19 vaccine, are unstable in the harsh environment of the gut, such as stomach acid. Additionally, because of their very large size, the highly efficient gut wall barrier severely limits the absorption of biologics into the bloodstream. Nucleic acid therapies therefore currently require administration by injection by a healthcare professional. Previous research efforts attempting to develop technologies for oral delivery of nucleic acids have not been successful.

Animal cells produce and release tiny particles (500-1000 times smaller than human hair width) called extracellular vesicles (EVs). These are membrane-bound particles and play a crucial role in cell-cell communication by transferring various biological molecule cargoes from one cell to another. This cargo also includes nucleic acids and proteins, making EVs ideal, naturally designed carriers to deliver these drugs across the gut wall. Previous research, including by our group, has shown that EVs present in cow milk are capable of efficiently crossing the gut wall. One could hence utilise these EVs, which are isolated from an abundant, inexpensive and sustainable source (milk) and to enable oral delivery of nucleic acids. However, the key challenge with the use of milk EVs to enable oral delivery of nucleic acids relates to their heterogenous nature (multiple particle types with different biological function) and the loading of large nucleic acids into membrane-bound EVs.

In this project we will identify the key components of milk EVs that drive intestinal permeation by screening the ability of these EVs to cross the gut wall. We will conduct this screening in laboratory models of the human gut wall (cells grown on plastic dishes). We will analyse the composition of EVs which cross the gut wall and compare it with those that do not have this ability. This will enable us to establish which EV components facilitate their transport across the gut wall. This information will at the same time enable us to selectively isolate EVs that cross the gut wall from a mixture of EVs present in milk. These EVs will then be engineered to enable drug (nucleic acid) loading. Engineered EVs will be tested in laboratory models of the human intestine, as well as animals for their efficacy for oral delivery of nucleic acids.

In addition to laboratory research detailed above, the project embeds knowledge-exchange activities (workshops, training and seminars) and will also establish an open access research facility for EV research at King's College London. This facility will house state-of-the-art equipment for studying EVs and will be available for free use to the research community working on EVs.

To deliver on the project's vision and overall objective, we have a strong, multidisciplinary team of researchers from London and Midlands institutions, namely King's College London, Aston University and the University of Nottingham. Additionally, we have incorporated collaboration with industry, specifically Micropore Technologies, who have capability to scale up the manufacturing of the new inexpensive but powerful EV-based therapies created in this project. The academic team will work closely together, as well as with the industrial partner, to deliver on different aspects of the project and the overall project objective, which is to create new medicines that would transform the management of many diseases, while being affordable and convenient for patients.

Oral administration of medicines is preferred by patients and is cost-effective for healthcare systems. Oral delivery of nucleic acids would have a transformative impact on therapies for local and systemic diseases. Current approaches investigated for oral nucleic acid delivery generally include the adoption of lipid-based nanoparticles, but their use is severely limited by poor gut stability and permeability.
Extracellular vesicles (EVs) are nanoscale vesicles released from most cells which play a crucial role in cell-cell communication via transfer of biomolecule cargoes. EVs are highly capable of crossing biological barriers. EVs present in bovine milk have been shown to maintain stability (including of cargo) in the gut's harsh chemical environment. We and others have also shown that milk EVs cross the intestinal epithelium with high efficacy. Milk EVs are therefore potentially ideal, safe, effective, inexpensive and scalable systems that could enable oral delivery of nucleic acids. But the key challenge relates to their heterogeneity and loading of macromolecules.

Drawing from the synergy of a multidisciplinary team with expertise in EVs, membrane proteins and nucleic acid delivery, the project will harness the biology of milk EVs through engineering biology approaches to transform these into inexpensive, oral nucleic acid therapies. This will be achieved by identifying key EV components that facilitate intestinal permeation (WP1) to enable selective isolation of epithelium-permeating EV subpopulations from milk (WP2). Epithelium-permeating EVs will then be engineered to achieve nucleic acid loading, in tandem with extensive testing of delivery performance in vitro and in vivo to enable optimisation (WP3). The project also incorporates community-building via knowledge-exchange activities and establishment of an open access research facility at King's providing free access to an advanced EV characterisation equipment for the EV engineering community.