Jonas Wallin
Senior lecturer, Director of third cycle studies, Department of Statistics
Single-cell quantification and dose-response of cytosolic siRNA delivery
Author
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