PhD defence by David Schultz

On Thursday, 22 August, David Schultz will defend his PhD thesis "Optimizing Quantification of mRNA-Lipid Nanoparticles and Elucidating Biodistribution after Device-Mediated Delivery in the Gastrointestinal Tract".

Time: 14:00
Place: Building 421, auditorium 74 & zoom:  https://dtudk.zoom.us/meeting/register/u5IpceypqT0iGtEx5ys_P4OWSpFnuj1ZuJx_
Please be aware that the PhD defense may be recorded - This will also be informed at the beginning of the PhD defense.

Supervisor: Professor Thomas Lars Andresen
Co-supervisor: Associate Professor Andrew Urquhart, DTU Health Tech

Assessment committee:
Professor Niels B Larsen, DTU Health Tech
Associate Professor Michael J. Mitchell, University of Pennsylvania
Professor Camilla Foged, Copenhagen University

Chairperson:
Senior Researcher Anders Elias Hansen, DTU Health Tech

Abstract:
The nucleic acid, mRNA, formulated in lipid nanoparticles (LNPs) has the potential to vaccinate i.e. against pathogens or cancer as well as act as a gene therapy.

When determining the concentration of mRNA inside the LNPs an assay using a fluorescent dye, the RiboGreen dye, is almost exclusively used in academia. Here, a surfactant is applied to release mRNA from LNPs to allow for detection by binding with the dye. Whilst the surfactant, Triton X-100, has been applied extensively, the effectiveness of the surfactant at releasing mRNA has not been elucidated or compared to other surfactants. Notably, Triton X-100 is of high concern to human health and the environment. The aim of this thesis was, therefore, to test whether three less harmful surfactants Zwittergent 3-14, Brij 93, and Tween 20 were suitable alternatives for releasing mRNA from LNPs. The project was the first to investigate whether other surfactants are on par with or better than Triton X-100 at releasing mRNA from LNPs. Key findings were that Tween 20 was better than Triton X-100 at releasing mRNA from LNPs at all concentrations for all formulations. Notably, Tween 20 was the only surfactant that was able to release mRNA consistently at all concentrations tested highlighting its potency. Furthermore, Tween 20 is on the Food and Drug Administration’s Generally Regarded as Safe (GRAS) list. This research contributes to more accurate mRNA concentration measurements, with which follow more precise dosing in animals i.e. more consistent and correct results.

When administering mRNA-LNPs to humans it is important to know to which organs the mRNA-LNPs will distribute in relation to the efficacy and safety profile. A novel administration route has recently been enabled by the innovation of ingestible devices that can inject directly into gastrointestinal tissue, e.g. stomach wall (SW) or intestinal wall (IW). Hence, the biodistribution after injection into SW and IW has yet to be elucidated. This project aimed to establish if mRNA-LNPs could be delivered using a microjet device whilst maintaining stability as well as to determine expression and biodistribution of mRNA in mice (as a well-tested animal research model) and minipigs (an animal model closer resembling human physiology). The project was the first to investigate mRNA expression and biodistribution after SW and IW injection and the first to conduct mRNA biodistribution studies in pigs. The key findings were that SW and IW injections lead to a broad distribution profile with a controlled release from the site of injection that is likely to be highly beneficial. Furthermore, SW injections in mice lead to expression levels on par with IM injections, which could indicate that SW injections could be used as an alternative to IM injections. Lastly, we show via histology that mRNA localizes in the germinal centers of the spleen after gastrointestinal injection, which for the CoVID-19 mRNA vaccination in humans resulted in affinity-matured long-term antibody responses. These results should help expedite the use and combination of mRNA-LNPs and ingestible devices for vaccinating against pathogens or potentially curing diseases.