Poster X16 - Genetically Engineered Extracellular Vesicles Retain Cargo Loading with Large-Scale Production

Introduction: Since the discovery that stem cell therapy efficacy is mediated in part by the release of paracrine factors, extracellular vesicles (EVs) have seen a surge in interest as a potentially safer and equally or more effective therapeutic. EVs are cell-secreted nanoparticles loaded with protein and nucleic acid cargo that are representative of their producer cells. This cargo provides EVs with bioactivity akin to their producer stem cells, without the tumorigenesis or storage concerns of cell therapies. Currently, clinical translation of EV therapeutics is limited by scalability and potency. Recent advances in EV production have included the development of perfusion bioreactors that decrease production materials, cost, and time, while increasing EVs produced per cell by over 80-fold. With the advancements in EV production techniques, potency has become the next hurdle to cross in translating EVs to the clinic. Genetic modification of the producer cells has shown promise in increasing EV yields, however, their compatibility with large-scale production methods have yet to be investigated.

Materials and

Methods: HEK293T cells were transfected with plasmids designed to overexpress either a miRNA or lncRNA of interest. Post-transfection, the cells were split into perfusion bioreactors as well as a T-175 cell culture flask and switched to EV depleted media. The media from the flasks were collected and replaced for two days while the bioreactors underwent 5 ml/min fluid flow with the media being collected after 24 hours. EVs were then isolated from the media using tangential flow filtration and measured using nanoparticle tracking analysis. RNA was then isolated from the EVs, and qPCR was performed to quantify respective loading between EVs from flask and bioreactor based production.

Results, Conclusions, and Discussions: Based on preliminary data, it is anticipated that EVs produced from genetically modified cells cultured in flasks and perfusion bioreactors will have similar specific RNA loading levels. Specifically, the loading of miR-146a and long non-coding RNA HOTAIR will have insignificant differences in EV loading between flask and bioreactor based production. These results are anticipated to confirm that genetically engineered EVs maintain their levels of cargo loading when production is scaled up using perfusion bioreactors. This would mark a significant step towards the clinical application of EV-based therapies, offering a scalable method to produce high yields of potent EVs. While there are many studies that have investigated the production of EVs from genetically modified producer cells, the culture method of said producer cells has remained in stagnant culture flasks. EV production per cell is significantly lower in these conditions compared to perfusion bioreactors which are required for

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