High-Throughput Barcoded Nanoparticles Predict Cationic Degradable Lipid-Like Materials for Pulmonary mRNA Delivery

Introduction:: Lipid nanoparticles (LNPs) delivering mRNA-based therapeutics holds immense potential for the treatment of a wide range of lung-associated diseases. However, the lack of effective methodologies capable of predicting the pulmonary delivery profile of chemically distinct lipid libraries poses a significant obstacle to the advancement of mRNA therapeutics. Here we report the implementation of a barcoded high-throughput screening system as a means to predict the lung targeting efficacy of cationic degradable (CAD) lipid-like materials. We demonstrate that our lead lung-specific CAD LNPs outperformed a gold-standard lung-tropic MC3/DOTAP LNP system for pulmonary mRNA delivery.

Materials and Methods:: A library of 180 novel CAD-Lipids were synthesized through “Schiff-base” reduction, formulated into CAD LNPs, and screened for luciferase mRNA delivery in HeLa cells. The selected CAD LNPs were used to encapsulate DNA barcodes (b-DNAs) and mRNA, pooled together, and systemically administered as a single pool into mice. DNA was extracted and b-DNA accumulation in each organ was then quantified using deep sequencing. The top-performing CAD LNPs delivering Cre mRNA to Ai14 mice were used to quantify transfected cell types in the lung.

Results, Conclusions, and Discussions:: Through in vitro high-throughput screening, we selected 96 CAD LNPs which effectively transfect HeLa cells. Then by analyzing the pooled 96 CAD LNPs and their tissue tropism using deep sequencing, we identified 21 CAD LNPs with lung-tropic properties. The top 4 CAD LNPs, formulated with luciferase mRNA, were further counter-screened in C57BL/6J mice, with LNP10 as the top-performing formulation, exhibiting luciferase expression predominantly in the lung (~90%). LNP10 was also found to preferentially deliver mRNA to lung endothelial cells at a clinically relevant dose of 0.3 mg/kg, which outperformed a gold standard lung-tropic MC3/DOTAP LNP system.
In conclusion, high-throughput barcoding technology can be utilized as efficient and predictive tools for identifying structurally distinct nanoparticles for extrahepatic delivery to the lung. We envision its potential applications to unlock mRNA delivery of newly designed lipid libraries for extrahepatic protein replacement, vaccine, and genetic disease treatments.


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