Amidine-Incorporated Degradable Lipids for Local, Systemic and Non-Liver mRNA Delivery

Introduction:: Lipid nanoparticles (LNPs) are the most clinically advanced non-viral vectors for mRNA delivery, in which cationic lipids greatly affect biodistribution, cellular uptake, endosomal escape and transfection efficiency. To discover efficacious cationic lipids and study structure-activity relationships (SARs), dozens or even hundreds of chemically divergent candidates are needed for comparison. However, the laborious synthesis of cationic lipids limits the process of discovery and investigation. There is great interest to develop new combinatorial chemistries to rapidly generate structurally diverse cationic lipids in order to study SARs for guiding the design of the next-generation delivery materials and fulfill the broader applications of mRNA therapeutics. We aim to develop new synthetic methods that can enable facile synthesis of degradable cationic lipids within hours, without requiring harsh conditions or specialized instruments.

Materials and Methods:: Amines, acrylates, Traut’s reagent and other reagents were obtained from commercial vendors. Amidine-Incorporated Degradable (AID) lipids were synthesized using a tandem multi-component reaction (T-MCR). Briefly, amine, Traut’s reagent, acrylate and triethylamine were dissolved in ethanol in a 1.5 mL Eppendorf tube. All tubes were put on a floating rack and sonicated in an ultrasonic cleaner for 1 h at room temperature and at a frequency of 40 kHz. mRNA-loaded LNPs were formulated by pipette/microfluidic mixing. High-throughput screening of LNPs was performed in HepG2 cells. Local or systemic mRNA delivery was performed in C57BL/6 mice.

Results, Conclusions, and Discussions:: We developed a one-pot T-MCR based on the rationally designed amine-thiol-acrylate conjugation, which enabled fast (1 h) and facile room-temperature synthesis of AID-lipids. SAR analysis of a combinatorial library of 100 chemically diverse AID-lipids led to the identification of a new tail-like amine–ring-alkyl aniline that generally affords efficacious lipids. Experimental and theoretical studies provided a new insight that the embedded bulk benzene ring could greatly enhance endosomal escape and mRNA delivery by aiding the lipid to adopt a more conical shape. The lead AID-lipid 12T-O14 aided both local SARS-CoV-2 spike mRNA and systemic fibroblast growth factor 21 mRNA delivery, which significantly outperformed a benchmark cationic lipid (DOTAP). Furthermore, 12T-O14 supplementation redirected liver-tropic LNPs to selectively target the lung or spleen, enabling CRISPR/Cas9-mediated gene editing in the lung and mRNA vaccine delivery to the spleen. Our synthetic methodology lowers the threshold and broadens the scope of MCR to develop cationic lipids within hours and requiring no specialized conditions. Moreover, AID-lipids hold great promise to aid the delivery of mRNA-based vaccines as well as protein replacement and gene editing therapeutics to target tissues.

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