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Endocytic Profiling of Cancer Cell Models Reveals Critical Factors Influencing LNP-Mediated mRNA Delivery and Protein Expression
- Source :
- Mol Ther
- Publication Year :
- 2019
- Publisher :
- Elsevier BV, 2019.
-
Abstract
- Lipid nanoparticles have great potential for delivering nucleic-acid-based therapeutics, but low efficiency limits their broad clinical translation. Differences in transfection capacity between in vitro models used for nanoparticle pre-clinical testing are poorly understood. To address this, using a clinically relevant lipid nanoparticle (LNP) delivering mRNA, we highlight specific endosomal characteristics in in vitro tumor models that impact protein expression. A 30-cell line LNP-mRNA transfection screen identified three cell lines having low, medium, and high transfection that correlated with protein expression when they were analyzed in tumor models. Endocytic profiling of these cell lines identified major differences in endolysosomal morphology, localization, endocytic uptake, trafficking, recycling, and endolysosomal pH, identified using a novel pH probe. High-transfecting cells showed rapid LNP uptake and trafficking through an organized endocytic pathway to lysosomes or rapid exocytosis. Low-transfecting cells demonstrated slower endosomal LNP trafficking to lysosomes and defective endocytic organization and acidification. Our data establish that efficient LNP-mRNA transfection relies on an early and narrow endosomal escape window prior to lysosomal sequestration and/or exocytosis. Endocytic profiling should form an important pre-clinical evaluation step for nucleic acid delivery systems to inform model selection and guide delivery-system design for improved clinical translation.
- Subjects :
- Endosome
Endocytic cycle
Fluorescent Antibody Technique
Gene Expression
Endosomes
Transfection
Endocytosis
Exocytosis
03 medical and health sciences
0302 clinical medicine
Genes, Reporter
Cell Line, Tumor
Drug Discovery
Genetics
Humans
RNA, Messenger
Molecular Biology
030304 developmental biology
Pharmacology
0303 health sciences
Messenger RNA
Chemistry
Hydrogen-Ion Concentration
Flow Cytometry
Lipids
Cell biology
Cell culture
030220 oncology & carcinogenesis
Cancer cell
Nanoparticles
Molecular Medicine
Original Article
Lysosomes
Subjects
Details
- ISSN :
- 15250016
- Volume :
- 27
- Database :
- OpenAIRE
- Journal :
- Molecular Therapy
- Accession number :
- edsair.doi.dedup.....cbb98452992fd2857b632619be0095af