Single-cell quantification and dose-response of cytosolic siRNA delivery
(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.
(Less)
- author
- Hedlund, Hampus
LU
; Du Rietz, Hampus
LU
; Johansson, Johanna M. LU ; Eriksson, Hanna C. LU
; Zedan, Wahed LU ; Huang, Linfeng ; Wallin, Jonas LU and Wittrup, Anders LU
- organization
-
- Tumor microenvironment
- LU Profile Area: Light and Materials
- Division of Molecular Medicine and Gene Therapy
- Radiation therapy
- LUCC: Lund University Cancer Centre
- Systemic Radiation Therapy Group (research group)
- Department of Statistics
- LTH Profile Area: Photon Science and Technology
- WCMM-Wallenberg Centre for Molecular Medicine
- publishing date
- 2023-12
- 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
- 2024-08-23 07:26:23
@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}}, }