Understanding the intracellular signalling pathways activated by lipid nanoparticle delivery of mRNA

Messenger RNA (mRNA) therapies rely on lipid nanoparticles (LNPs) as delivery vehicles to transfect exogenously transcribed mRNA into cells for prophylactic or therapeutic use. LNP delivered mRNA (LNP/mRNA) in the clinic can induce a transient, innate immune response resulting in local and systemic inflammation, which is a tolerability issue for therapeutic applications. The mechanisms underlying the inflammatory response and key triggers of innate immune signalling remain undefined. Therefore, the aim of this research was to investigate whether LNP/mRNA induces inflammation through Toll Like Receptors (TLRs) and whether endoplasmic reticulum (ER) stress is activated due to additional protein translation of the delivered mRNA. To address this aim, human dermal fibroblasts (NHDFs) were identified as a translationally relevant *in vitro* model with the capacity to express the transfected mRNA and release inflammatory cytokines. Using the NHDFs and small molecule inhibitors, TLR3 and TLR4 were identified to have roles in the inflammatory response induced by LNP/mRNA. TLR3 inhibition also improved the levels of protein translated from the delivered mRNA, highlighting the adverse effect of TLR signalling on the therapeutic efficacy of mRNA therapies. Furthermore, CRISPR gene editing technology was deployed to delete expression of MYD88 and TICAM1, downstream adaptor proteins of TLRs, where deletion of MYD88 reduced the LNP/mRNA induced cytokine response suggesting that TLR signalling has a role in LNP/mRNA induced inflammation. Subsequently, the impact of LNP/mRNA on the ER was assessed by measuring markers of the unfolded protein response (UPR) with the use of reporter cell lines and quantitative PCR analysis. LNP/mRNA induced the UPR independently of the expressed protein. Furthermore, UPR activation occurred at least 24 h post transfection, which coincided with observed cytotoxicity, suggesting that the UPR could be activated indirectly by other stress pathways activated in the cell. During investigations, the expression of LNP/mRNA displayed a bell-shaped dose-response curve *in vitro*, and cytotoxicity was noted across multiple cell types at certain LNP/mRNA concentrations. The mechanisms underlying these observations were further interrogated using fluorescent imaging techniques and showed that the bell-shaped dose-response curves are the result of insufficient cellular uptake of the LNP/mRNA at higher concentrations. The data show that the loss of expression at higher LNP/mRNA concentrations is rescued if the amount of available serum protein increases. The data indicate that the cellular uptake of LNPs depends on a dynamic relationship between the LNP concentration and protein corona. This relationship should be considered for LNP applications to derive optimal efficiency. Finally, extensive testing of different LNP components and inhibitors of endosomal progression demonstrated that cytotoxicity is driven by the LNP rather than the mRNA cargo, with a clear correlation between LNP-induced endosomal disruption and cytotoxicity established. Overall, this project provides direction on the cellular pathways that could influence the efficacy and inflammatory responses induced by LNP/mRNA and describes a novel correlation linking endosomal disruption with cytotoxicity. The cell stress reported *in vitro* may be linked to inflammation in the clinic and contribute to the growing evidence of the adjuvant nature of LNPs. Understanding these pathways can support the development of mitigation strategies to improve the safety of mRNA therapies, thereby enabling their broader application in therapeutic contexts, where inflammatory responses are unwanted.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Bates, Stephanie

Advisor dc:contributor.advisor

• Gay, Nicholas

• Endosomal disruption
• Lipid nanoparticles
• mRNA therapy

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ee123ade-33a3-4625-9544-c60baa7651a0/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.112923

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/375131