LNP formulations for efficient hepatic delivery studied with an in-vivo endosomal escape assay

ABSTRACT: Endosomal escape is a central barrier to efficient nucleic acid delivery by lipid nanoparticles (LNPs) and remains challenging to quantify in vivo. We report a library of branched ionizable phospholipids (BiPs) that markedly enhance mRNA delivery to the liver. The lead candidate BiP-20 outperformed the clinical benchmark LP01 by 4-fold for CRISPR–Cas9 editing of the TTR gene at low dose with rapid pharmacokinetics. To quantify the endosomal escape kinetics of BiP-20, we used LysoTag mice, which allow immunoisolation of liver lysosomes, and our Lysosomal Barcoding method, finding that ~9% of BiP-20 LNPs reach the cytosol within 30 minutes of administration. Lysosomal proteomics revealed mechanistic regulators of escape and BiP-20–induced alterations in endosomal maturation and recycling pathways. Loss of Rab7, a mediator of late endosomal maturation, increased LNP escape. These findings provide a potent class of ionizable lipids for RNA delivery, a method to quantify endosomal escape in vivo, and mechanistic insight into the endolysosomal determinants of LNP trafficking.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Liver

SUBMITTER: Phillip Wilmarth  

LAB HEAD: Gaurav Sahay

PROVIDER: PXD072066 | Pride | 2026-01-20

REPOSITORIES: Pride