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Jonas Wallin. Photo.

Jonas Wallin

Senior lecturer, Director of third cycle studies, Department of Statistics

Jonas Wallin. Photo.

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

Author

  • Hampus Hedlund
  • Hampus Du Rietz
  • Johanna M. Johansson
  • Hanna C. Eriksson
  • Wahed Zedan
  • Linfeng Huang
  • Jonas Wallin
  • Anders Wittrup

Summary, in English

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.

Department/s

  • Tumor microenvironment
  • LU Profile Area: Light and Materials
  • Division of Molecular Medicine and Gene Therapy
  • Systemic radiation therapy
  • LUCC: Lund University Cancer Centre
  • Systemic Radiation Therapy Group
  • Department of Statistics
  • LTH Profile Area: Photon Science and Technology
  • WCMM-Wallenberg Centre for Molecular Medicine

Publishing year

2023-12

Language

English

Publication/Series

Nature Communications

Volume

14

Issue

1

Document type

Journal article

Publisher

Nature Publishing Group

Topic

  • Cell and Molecular Biology

Status

Published

Project

  • Tracking the subcellular fate of therapeutic RNAs

Research group

  • Systemic Radiation Therapy Group

ISBN/ISSN/Other

  • ISSN: 2041-1723