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Single-cell quantification and dose-response of cytosolic siRNA delivery

Hedlund, Hampus LU ; Du Rietz, Hampus LU orcid ; Johansson, Johanna M. LU ; Eriksson, Hanna C. LU orcid ; Zedan, Wahed LU ; Huang, Linfeng ; Wallin, Jonas LU and Wittrup, Anders LU (2023) In Nature Communications 14(1).
Abstract

Endosomal escape and subsequent cytosolic delivery of small interfering RNA (siRNA) therapeutics is believed to be highly inefficient. Since it has not been possible to quantify cytosolic amounts of delivered siRNA at therapeutic doses, determining delivery bottlenecks and total efficiency has been difficult. Here, we present a confocal microscopy-based method to quantify cytosolic delivery of fluorescently labeled siRNA during lipid-mediated delivery. This method enables detection and quantification of sub-nanomolar cytosolic siRNA release amounts from individual release events with measures of quantitation confidence for each event. Single-cell kinetics of siRNA-mediated knockdown in cells expressing destabilized eGFP unveiled a... (More)

Endosomal escape and subsequent cytosolic delivery of small interfering RNA (siRNA) therapeutics is believed to be highly inefficient. Since it has not been possible to quantify cytosolic amounts of delivered siRNA at therapeutic doses, determining delivery bottlenecks and total efficiency has been difficult. Here, we present a confocal microscopy-based method to quantify cytosolic delivery of fluorescently labeled siRNA during lipid-mediated delivery. This method enables detection and quantification of sub-nanomolar cytosolic siRNA release amounts from individual release events with measures of quantitation confidence for each event. Single-cell kinetics of siRNA-mediated knockdown in cells expressing destabilized eGFP unveiled a dose-response relationship with respect to knockdown induction, depth and duration in the range from several hundred to thousands of cytosolic siRNA molecules. Accurate quantification of cytosolic siRNA, and the establishment of the intracellular dose-response relationships, will aid the development and characterization of novel delivery strategies for nucleic acid therapeutics.

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author
; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Nature Communications
volume
14
issue
1
article number
1075
publisher
Nature Publishing Group
external identifiers
  • scopus:85149054370
  • pmid:36841822
ISSN
2041-1723
DOI
10.1038/s41467-023-36752-1
project
Tracking the subcellular fate of therapeutic RNAs
language
English
LU publication?
yes
id
a46108b2-415c-4c4d-9ce9-58c8bd712ab0
date added to LUP
2023-03-13 11:27:06
date last changed
2024-12-13 20:50:10
@article{a46108b2-415c-4c4d-9ce9-58c8bd712ab0,
  abstract     = {{<p>Endosomal escape and subsequent cytosolic delivery of small interfering RNA (siRNA) therapeutics is believed to be highly inefficient. Since it has not been possible to quantify cytosolic amounts of delivered siRNA at therapeutic doses, determining delivery bottlenecks and total efficiency has been difficult. Here, we present a confocal microscopy-based method to quantify cytosolic delivery of fluorescently labeled siRNA during lipid-mediated delivery. This method enables detection and quantification of sub-nanomolar cytosolic siRNA release amounts from individual release events with measures of quantitation confidence for each event. Single-cell kinetics of siRNA-mediated knockdown in cells expressing destabilized eGFP unveiled a dose-response relationship with respect to knockdown induction, depth and duration in the range from several hundred to thousands of cytosolic siRNA molecules. Accurate quantification of cytosolic siRNA, and the establishment of the intracellular dose-response relationships, will aid the development and characterization of novel delivery strategies for nucleic acid therapeutics.</p>}},
  author       = {{Hedlund, Hampus and Du Rietz, Hampus and Johansson, Johanna M. and Eriksson, Hanna C. and Zedan, Wahed and Huang, Linfeng and Wallin, Jonas and Wittrup, Anders}},
  issn         = {{2041-1723}},
  language     = {{eng}},
  number       = {{1}},
  publisher    = {{Nature Publishing Group}},
  series       = {{Nature Communications}},
  title        = {{Single-cell quantification and dose-response of cytosolic siRNA delivery}},
  url          = {{http://dx.doi.org/10.1038/s41467-023-36752-1}},
  doi          = {{10.1038/s41467-023-36752-1}},
  volume       = {{14}},
  year         = {{2023}},
}