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<p>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 <i>TTR</i> 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.</p>

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

  • 期刊论文

  • 发表日期：

    2026年3月11日

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  • Antony Jozić,
  • Chloé Le Roux,
  • Jeonghwan Kim,
  • Mathieu Berchel,
  • Deepak Kumar Sahel,
  • Emily K. Bodi,
  • Michelle Palumbo,
  • Aishwarya Vasudevan,
  • Namratha Turuvekere Vittala Murthy,
  • Yulia Eygeris,
  • Milan Gautam,
  • Elissa Bloom,
  • Anthony P. Barnes,
  • Paul-Alain Jaffrès,
  • Gaurav Sahay

摘要

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.