Molecular characterization of RNA delivery: how to escape endosomal compartments?

• Applicant: Emeline Bonsergent, Ph.D.
• Subject Area: Cell Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 525131893

Macromolecule delivery is used for both research and therapeutic purposes. Even though some applications, such as COVID19 vaccines, are efficient, there is still a knowledge gap to truly understand the molecular mechanisms underlying the delivery of molecule like RNA. In my project proposal, I aim to use an interdisciplinary approach to study the mechanism of endocytic trafficking and endosomal escape of mRNA packaged into lipid nanoparticles (LNPs). Delivery is a multistep process whereby mRNA formulated in formulated in LNPs must be taken up by cells and sorted into specific endosomal compartments, unpackaged from the LNP, and escape the endosome into the cytosol. Endocytosis and endosomal escape are the key limiting steps in mRNA delivery, causing poor efficiency and high toxicity, and limiting its applications beyond vaccines. Despite their importance, these mechanisms are poorly understood. Here, I will take advantage of the unique expertise of the host group in endocytosis and biochemical reconstitution to pursue four successive objectives. (1) I will quantitatively characterize the intracellular trafficking of both LNP vehicle (lipid) and cargo (mRNA), combining biochemical assays and imaging of modified lipids probes.(2) To improve the characterization of endocytic compartments, I will sequentially immuno-isolate each endosomal subpopulation and analyze their proteome and lipidome using mass spectrometry. (3) I will develop an in vitro assay of endosomal escape, using isolated endosomes containing LNP-mRNA, to determine the most favorable compartments for the mRNA escape to occur. (4) Finally, based on Aims1-3, I will characterize the membrane dynamics involved in the escape, quantifying the lipid interactions occurring during this process. I expect this project to generate important insights into the molecular mechanisms of endosomal trafficking and escape, as pre-requisite to improve therapeutic delivery while decreasing cyto-toxicity.

• DFG Programme: WBP Position