(414f) Two Nitrogen-Based Ionizable Lipid System for mRNA Delivery to T Lymphocytes

Delivery systems for mRNA-based therapeutics have gained enormous attention since the FDA approval of lipid nanoparticle (LNP)-based vaccines for SARS-CoV-2. LNPs are the most clinically advanced platform for nucleic acid delivery and can be formulated for various applications such as gene editing, protein replacement, and vaccine applications. Despite the enormous potential of these delivery systems, questions remain about the repeated dosage of LNPs and their potential side effects.

To address these concerns, we designed a two-nitrogen library of lipids. It was hypothesized that, at the same lipid-tertiary-amine to mRNA-phosphate (N/P) ratio, the two nitrogen atoms in the lipid would be utilized to encapsulate mRNA at a relatively low molecular weight, which in turn could reduce the total lipid dosage compared to a one-nitrogen lipid. This class of lipids could potentially mitigate the side effects posed by single-nitrogen lipid while maintaining the same performance level. We screened the library of lipids against hard-to-transfect Jurkat human T cells in vitro. We found that three lead LNPs, distinct in structure, performed well. The LNPs were optimized by evaluating the effect of cholesterol and its analogues. Furthermore, the cell uptake and endolytic pathways were determined for the lead LNPs. The three lead LNPs were then tested in vivo using Balb/c mice. Lipid 1 LNPs showed translation efficiency in the liver while Lipid 2 LNPs showed higher protein translation in spleen relative to SM-102-based LNP. Overall, the library of lipids showed promising results for mRNA delivery with reduced total lipid dosage.