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Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown
Open accessAbstract
A central hurdle in developing small interfering RNAs (siRNAs) as therapeutics is the inefficiency of their delivery across the plasma and endosomal membranes to the cytosol, where they interact with the RNA interference machinery. With the aim of improving endosomal release, a poorly understood and inefficient process, we studied the uptake and cytosolic release of siRNAs, formulated in lipoplexes or lipid nanoparticles, by live-cell imaging and correlated it with knockdown of a target GFP reporter. siRNA release occurred invariably from maturing endosomes within ~5-15 min of endocytosis. Cytosolic galectins immediately recognized the damaged endosome and targeted it for autophagy. However, inhibiting autophagy did not enhance cytosolic siRNA release. Gene knockdown occurred within a few hours of release and required <2,000 copies of cytosolic siRNAs. The ability to detect cytosolic release of siRNAs and understand how it is regulated will facilitate the development of rational strategies for improving the cytosolic delivery of candidate drugs.
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“∼2000 cytoplasmic siRNAs are estimated to achieve maximal silencing, whereas ∼50 000 are required for an ASO [54, 55].”
From Elucidating brain transport pathways and cell type-dependent gene silencing of a durable lipid–siRNA conjugate administered into cerebrospinal fluid (2025) · cites this paper as [54]
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“Indeed, only a small amount of lipid nanoparticles may be able to escape from the endosome137–139.”
From Lipid nanoparticles for mRNA delivery (2021) · cites this paper as [139]
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“Following cellular internalization, lipid nanoparticles are usually trapped in endosomal compartments137–139.”
From Lipid nanoparticles for mRNA delivery (2021) · cites this paper as [139]
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Metadata
- Authors
- Wittrup, A., Ai, A., Liu, X., Hamar, P., Trifonova, R., Charisse, K., Manoharan, M., Kirchhausen, T., & Lieberman, J.
- Affiliations
- Boston Children's Hospital (US)
- Alnylam Pharmaceuticals (United States) (US)
- Corresponding author
- Wittrup, Anders
- Lieberman, Judy
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2015-07-20
- Volume
- 33
- Issue
- 8
- Pages
- 870-876
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC4663660
- OpenAlex
- W1844274376
- Cited by
- 693
- References
- 30
- Citations by year
- 2026: 101 · 2025: 104 · 2024: 82 · 2023: 60 · 2022: 59 · 2021: 63 · 2020: 53 · 2019: 40 · 2018: 54 · 2017: 37 · 2016: 31 · 2015: 8
- FWCI
- 18.70
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Lund University Publications (Lund University) (submittedVersion)
- Funders
- National Institutes of Health
- National Cancer Institute (R01CA139444, CA139444)
- National Institute of General Medical Sciences (GM075252, R01GM075252)
- National Institute of Allergy and Infectious Diseases (AI090671, AI057159, R01AI090671, U54AI057159)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Keywords
- siRNA delivery, endosomal escape, lipoplexes, lipid nanoparticles, endocytosis, autophagy, galectins, gene knockdown, live-cell imaging, RNA interference, siRNA therapeutics
- MeSH terms
- HeLa Cells, Humans, Lipids, Luminescent Proteins, Endosomes, RNA Interference, RNA, Small Interfering, Nanoparticles, Gene Knockdown Techniques
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