In vivo endosomal escape assay identifies mechanisms for efficient hepatic LNP delivery

Mar 12, 2026Nature biotechnology

Live test finds how liver cells efficiently receive lipid nanoparticle delivery

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CRISPR Gene Editing on OpenScience ↗PubMed ↗DOI ↗

Abstract

BiP-20 outperformed the clinical benchmark LP01 by eightfold for CRISPR-Cas9 editing of the TTR gene at low dose.

A library of branched ionizable phospholipids was developed to improve messenger RNA delivery to the liver.
Approximately 8% of BiP-20 lipid nanoparticles reach the cytosol within 30 minutes of administration.
Lysosomal proteomics identified key regulators of endosomal escape and changes in endosomal maturation and recycling pathways induced by BiP-20.
The loss of Rab7, which is involved in late endosomal maturation, was associated with increased lipid nanoparticle escape.

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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 that markedly enhance messenger RNA delivery to the liver. The lead candidate BiP-20 outperformed the clinical benchmark LP01 by eightfold 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 ~8% of BiP-20 LNPs reach the cytosol within 30 min 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.