(M1530-12-83) Effect of LNP Components on mRNA-Mediated Protein Expression of Lipid Nanoparticles in Different Organs

Purpose: In recent years, RNA therapeutics have received tremendous attention. To fully realize the potential of RNA therapeutics, lipid nanoparticles (LNPs) have been developed as an efficient delivery system. While LNPs have been widely used for delivery of RNA therapeutics into liver, non-hepatic delivery remains highly challenging. To better understand the mRNA expression of LNPs in different organs, we have evaluated the translation of luciferase encoded mRNA using various LNP systems.

Methods: LNP formulations were manufactured using NanoAssemblr® Ignite with different compositions with Luciferase encoded mRNA. Size and PDI were determined using Dynamic Light Scattering. The encapsulation efficiency was determined using the Ribogreen™ assay. The pKa of mRNA LNPs were measured by TNS assay. Luciferase expression as total flux in various organs were evaluated in vivo and ex vivo using IVIS imaging. Briefly, CD-1 mice were dosed with luciferase encode mRNA LNP intravenously at a 0.1 mg/kg dose. Four hours later mice were injected with D-luciferin and imaged using IVIS. After euthanasia, different organs were dissected for ex-vivo luminescence imaging using IVIS.

Results: Size of the LNP was reported to be between 60-150 nm with PDI < 0.2. The experimental pKa of different ionizable lipids were between 4.9 to 7.4. All LNPs tested demonstrated high luciferase protein expression in the liver. Liver expression levels were comparable to other clinically demonstrated lipids.

Conclusion: For ionizable lipids, there is a correlation between overall protein expression and pKa of ionizable lipids. We have also seen that various components in the LNP compositions have implications with respect to protein expression in liver, spleen, and other organs. These parameters affect the biodistribution and translation of the lipid nanoparticles.