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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

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			Action	DRS
	2024.06_UP000000589_10090_Mus_musculus_canonical_both.fasta	Fasta
	DE-candidate_summary.xlsx	Xlsx
	File_list.xlsx	Xlsx
	GAUR-1370_KO-results_edgeR-exact.txt	Txt
	GAUR-1370_KO-treatments.xlsx	Xlsx

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Project description:Cocaine-mediated repression of the histone methyltransferase (HMT) G9a has recently been implicated in transcriptional, morphological, and behavioral responses to chronic cocaine administration. Here, using a ribosomal affinity purification approach, we find that G9a repression by cocaine occurs in both Drd1 (striatonigral)- and Drd2 (striatopallidal)-expressing medium spiny neurons (MSNs). Conditional knockout and overexpression of G9a within these distinct cell types, however, reveals divergent behavioral phenotypes in response to repeated cocaine treatment. Our studies further indicate that such developmental deletion of G9a selectively in Drd2 neurons results in the unsilencing of transcriptional programs normally specific to striatonigral neurons, and the acquisition of Drd1-associated projection and electrophysiological properties. This partial striatopallidal to striatonigral ‘switching’ phenotype in mice indicates a novel role for G9a in contributing to neuronal subtype identity, and suggests a critical function for cell-type specific histone methylation patterns in the regulation of behavioral responses to environmental stimuli. Polyribosome associated mRNAs from 2-5 month old, age and sex matched Drd1-Cre; Drd1-TRAP; G9afl/fl and Drd1-TRAP; G9afl/fl, Drd2-Cre; Drd2-TRAP; G9afl/fl and Drd2-TRAP; G9afl/fl mice (n = 2-4 mice/genotype/drug treatment, 2 hours after the last of eight repeated cocaine injections of 20 mg/kg/day) were obtained as previously described. EGFP labeled ribosomes and associated mRNAs were immunoprecipitated using a mix of two monoclonal anti-GFP antibodies (50 μg of clones #19C8 and #19F7 for each IP, available at Sloan-Kettering Monoclonal Antibody Facility). Purified mRNA was amplified and processed for microarray and qPCR analysis using the Affymetrix two-cycle cDNA Synthesis kit (Affymetrix) as previously described. Affymetrix Mouse Genome 430 2.0 arrays were used in all experiments. Information regarding the array design and features can be found at www.affymetrix.com. Mouse Genome 430 2.0 arrays were scanned using the GeneChip Scanner 3000 (Affymetrix) and globally scaled to 150 using the Affymetrix GeneChip Operating Software (GCOS v1.4).

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

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