High-Throughput In Vivo Screening Identifies Structural Factors Driving mRNA Lipid Nanoparticle Delivery to the Brain

Abstract

Achieving systemic nonviral delivery of large nucleic acids such as mRNA to the brain is challenging due to high off-target delivery and the blood-brain barrier (BBB), a cellular barrier which prevents most nucleic acids in circulation from entering the brain. Ionizable lipid nanoparticles (LNPs) are a promising class of nanocarriers to facilitate the delivery of mRNA, as their highly modular nature enables fine-tuning of the LNP formulation for targeted delivery applications. In this work, we explore the role of ionizable lipid chemical structure and lipid molar ratios within the LNP formulation on mRNA delivery to and transfection of the brain. We utilize a high-throughput in vivo screening approach based on mRNA barcoding to study a large library of LNPs made with systematically varied ionizable lipid structures, amounts of ionizable lipid, and amounts of lipid-polyethylene glycol (PEG). We find that ionizable lipids with longer tail structures and linear amine cores can facilitate mRNA delivery to the mouse brain, and ultimately identify a specific ionizable lipid, C14-306, that facilitates brain transfection coupled with reduced liver transfection compared to an FDA-approved benchmark formulation. Furthermore, the lead LNP formulated with C14-306 is able to increase neuronal transfection and facilitate Cre-mediated recombination in the brain. Finally, safety analyses demonstrate that the lead LNP does not induce BBB leakage, increases in serum inflammatory cytokine levels, or increases in serum liver enzyme levels. Overall, our work highlights the utility of molecular barcoding for high-throughput screening of LNPs for delivery to the brain and suggests several design principles to guide the engineering of next-generation brain-tropic LNPs.