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

Hampus Hedlund, Hampus Rietz, Johanna M. Johansson, Hanna C. Eriksson, Wahed Zedan, Linfeng Huang, Jonas Wallin and Anders Wittrup ()
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Hampus Hedlund: Lund University
Hampus Rietz: Lund University
Johanna M. Johansson: Lund University
Hanna C. Eriksson: Lund University
Wahed Zedan: Lund University
Linfeng Huang: Duke Kunshan University
Jonas Wallin: Lund University
Anders Wittrup: Lund University

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: 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 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.

Date: 2023
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DOI: 10.1038/s41467-023-36752-1

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