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In vivo endosomal escape assay identifies mechanisms for efficient hepatic LNP delivery
Antony Jozić1,2, Chloé Le Roux3, Jeonghwan Kim4
1Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Portland, OR, USA.
Nature Biotechnology
|March 12, 2026
Summary
New ionizable lipids significantly improve messenger RNA delivery via lipid nanoparticles (LNPs) to the liver. A lead candidate, BiP-20, demonstrated superior performance for gene editing, with a novel method quantifying rapid endosomal escape.
Area of Science:
- Biotechnology
- Molecular Biology
- Drug Delivery Systems
Background:
- Efficient nucleic acid delivery using lipid nanoparticles (LNPs) is hindered by endosomal escape, a critical and difficult-to-quantify barrier in vivo.
- Understanding and overcoming endosomal entrapment is essential for advancing LNP-based therapeutics.
Purpose of the Study:
- To develop and characterize novel branched ionizable phospholipids for enhanced messenger RNA (mRNA) delivery via LNPs.
- To quantify the in vivo endosomal escape kinetics of a lead LNP candidate (BiP-20).
- To elucidate the mechanistic basis of LNP trafficking and endosomal escape.
Main Methods:
- Synthesis and screening of a library of branched ionizable phospholipids for LNP formulation.
- In vivo evaluation of LNP-mediated mRNA delivery and gene editing efficiency (CRISPR-Cas9 TTR gene editing) in mice.
- Quantification of endosomal escape using LysoTag mice and the Lysosomal Barcoding method.
- Lysosomal proteomics to identify regulators of LNP trafficking and endosomal escape.
Main Results:
- A novel class of branched ionizable lipids was identified, significantly enhancing liver mRNA delivery.
- The lead candidate, BiP-20, outperformed the clinical benchmark LP01 eightfold for TTR gene editing at low doses with rapid pharmacokinetics.
- ~8% of BiP-20 LNPs achieved cytosolic delivery within 30 minutes, as quantified by the Lysosomal Barcoding method.
- Lysosomal proteomics revealed that Rab7, a key regulator of endosomal maturation, influences LNP escape, with its loss enhancing escape.
Conclusions:
- Branched ionizable phospholipids represent a potent class of lipids for improving LNP-based RNA delivery.
- A robust method for quantifying in vivo endosomal escape kinetics was established.
- Mechanistic insights into the role of endolysosomal pathways, particularly Rab7, in LNP trafficking and escape were gained.
