Mesenchymal stem cell-derived extracellular vesicles as a cell-free therapy for ARDS.

Acute Respiratory Distress Syndrome (ARDS) is a severe clinical syndrom that affects 20% of all critically ill patients in intensive care. Unfortunately, around 30-50% of these people will die from the condition and the quality of life of the survivers can be seriously reduced due to the consequent chronic lung problems.There is no effective treatments for this condition at this time and therefore new medicines are urgently needed. ARDS is characterised by an excessive and disregulated inflammation in the lung that leads to inappropriate infiltration of immune cells, lungs fill with water and fail to maintain its main function - breathing. In order to breath these patients require mechanical ventilation.
Recently more and more clinicians and basic scientists attention is drawn to cell-based therapies. One of the most promising candidate for a cell based therapy are cells termed Mesenchymal Stem Cells (MSCs), which represent one kind of adult stem cells (can be easily isolated from tissues of adult patients or healthy volunteers), because of this, there is no ethical issues. MSCs could easily be propagated and manipulated under laboratory conditions and have capacity to produce multiple mediators with immuno-modulatory properties. However, concerns regarding safety of long-term effects of MSC administration (e.g. development of tumors) as well as logistical challenges may limit their successful clinical translation. Accumulating evidence now suggests that MSCs act primarily through secretion of different biologically active mediators. Therefore researchers are increasingly considering MSC cell products (which can not replicate and therefore form tumors) as an alternative to the whole cell therapy.
In this project we will investigate the therapeutic potential of small membrane wrapped "exracellular vesicles" released by MSCs. These vesicles represent the key component of MSCs secretome and are relatively easy to isolate from MSC conditioned medium by high speed centrifugation. Previously we and others had demonstrated that these vesicles were capable to recapitulate therapeutic effects of MSCs in various pre-clinical models of ARDS. Furthemore, in our recent work we had found that these vesicles contain organelles responsible for maintaining cell bioenergetics (mitochondria). This is very exciting finding as it is known that mitochondrial function is impaired in acute inflammatory conditions such as ARDS.
In this project we will use small animal model of ARDS to investigate the therapeutic potential of these vesicles, optimise best route and strategy of administration, gain isights into their bio-distribution in the tissues and also investigate whether or not functionally active mitochondria are important for the therapeutic effect and which tissues are major recipients of the mitochondria transferred in the EVs. These studies will be further complemented by the investigation of the EV mitochndrial transfer to primary human lung cells in vitro. These data are an essential requirement by regulatory bodies such as UK Medicines and Healthcare Products Regulatory Agency and European Medicines Agency for clincal translation of new therapies. The principle anticipated output of this project will be the establishment of a defined strategy for clinical development and knowledge of the specific mechanism of action (based on mitochondrial transfer) of the MSC derived extracellular vesicles. More immediately these data will inform further pre-clinical studies in larger animal models of ARDS required to obtain neccessary data on safety, efficacy and mechanism of action of MSCs EVs. Collectively data in small and large animal models will inform early phase clinical trials for MSC extracellular vesicles in patients with ARDS.

Acute respiratory distress syndrome (ARDS) is a major cause of mortality in the critically ill patients with no effective pharmacological treatment. Mesenchymal Stem Cells (MSCs) hold significant therapeutic promise for this condition and are rapidly progressing to clinical trials across the world. However, concern for the use of stem cells, specifically the risk of iatrogenic tumor formation, remains unresolved. Exracellular vesicles are key components of the MSC secetome and were reported to recapitulate efect of MSCs in the pre-clinical models of ARDS. We have demonstrated that MSC EVs are central for MSC effect on modulation of macrophages in vivo and in vitro. Furthemore we have identified that functionally active mitochondria secreted in EVs are largely responsible for the EVs effects. In this proposal we wish to undertake feasibility studies which will lead to further focussed pre-clinical development of MSC EV-based therapy for ARDS through the MRC DPFS scheme and utimately to Phase I/II clinical trial within the next 5 years.
The main goal of this proposal is to evaluate therapeutic potential of MSC EVs and investigate their mechanism of action through mitochondrial transfer in the clinically relevant in vitro and in vivo models of ARDS. We will address the following research questions:
1) To determine the efficacy of intravenous and intra-tracheal EVs administration routes;
2) To assess the efficacy of repeated EV administration vs. single dose;
3) To find the optimal therapeutic dose;
4) To establish comparative efficacy of the frozen-thawed vs. freshly isolated EVs;
5) To investigate the mechanism of EVs action via mitochondrial transfer.
To answer these questions we will use murine model of E.coli-induced lung injury and primary human lung epithelial, endothelial and immune cells stimulated by pro-inflammatory cytokines.
These data are essential requirement to unlock the clincal development by the regulatory bodies such as MHRA and EMA.

The mortality associated with ARDS remains high, between 30-40%. It is a common condition affecting all age groups with an estimated incidence of 79 per 100, 000 patient years. ARDS has significant resource implications, prolonging intensive care unit (ICU) and hospital stay, and requiring rehabilitation in the community. The cost per ICU bed-day in the UK exceeds £1800 and delivery of critical care to patients with ARDS accounts for a significant proportion of ICU capacity. Only 54% of survivors are able to return to work 12 months after hospital discharge. The high incidence, mortality, longterm consequences and economic costs mean that ARDS is an extremely important problem.
Results from this work can be easily translated into the development of new optimised therapy based on the MSC derived extracellular vesicles, which would make a more uniform and predictable product compared to whole cell administration. In the long-term this research will have implications to improve health and wellbeing in the UK and globally. Hastening the recovery from ARDS would lead to a shorter ICU stay, with attendant reductions in nosocomial infection, and other critical illness complications including neuropathy/myopathy/ delirium. This would have direct short term benefits to the patients themselves, including reduced hospital stay and complications, leading hopefully to a better long term functional outcome, with more patients returning to work. Additionally this would lead to a reduced burden for carers and reduced cost of stay and long term healthcare provision.
Understanding the mechanisms mediating effect of MSC EVs in ARDS, specifically mitochondrial transfer will lead to identification of other therapeutic targets with the potential for further development as a pharmacological treatment for ARDS.
In addition to this condition, the understanding of mechanisms of EV mediated mitochondrial transfer will open new avenues of research for other diseases mediated by impaired mitochondrial function, both in the lung and in other organs.
With this in mind, this research is likely to impact other researchers both academically and industrially in development of follow-on therapeutics and biomaterials that can exploit these effects. Therefore it is tangible that if successful, this project could lead on to the development of other daughter therapeutics that could not only help patients suffering other inflammatory diseases, but also generate new R & D jobs and revenue for the UK.
This proposal is at the forefront of rapidly emerging MSC field and of global competitiveness. Results from this research will be published in high impact journals and presented at the top international and national conferences. This will gain further recognition of the QUB as an internationally competitive University contributing to the reputation of the UK as a world leader in the biosciences.
Results from this project have the potential to attract R&D investment from biotechnology and pharmaceutical companies (e.g. through development of the potency assays important in the cell manufacturing process or may provide new insightsinto the cell or extracellular vesicles manipulation in order to improve efficacy). Any commercialisation would increase wealth and foster economic competitiveness in the UK.
Furthermore, MSC-derived EVs have a great potential to be developed into therapeutic for a wide range of conditions beyond ARDS.