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Imaging small molecule-induced endosomal escape of siRNA

Du Rietz, Hampus LU orcid ; Hedlund, Hampus LU ; Wilhelmson, Sten LU ; Nordenfelt, Pontus LU orcid and Wittrup, Anders LU (2020) In Nature Communications 11(1).
Abstract

Small interfering RNAs (siRNAs) are a new class of promising therapeutic molecules that can be used for sequence-specific downregulation of disease-causing genes. However, endosomal entrapment of siRNA is a key hurdle for most delivery strategies, limiting the therapeutic effect. Here, we use live-cell microscopy and cytosolic galectin-9 as a sensor of membrane damage, to probe fundamental properties of endosomal escape of cholesterol-conjugated siRNA induced by endosome-disrupting compounds. We demonstrate efficient release of ligand-conjugated siRNA from vesicles damaged by small molecules, enhancing target knockdown up to ∼47-fold in tumor cells. Still, mismatch between siRNA-containing and drug-targeted endolysosomal compartments... (More)

Small interfering RNAs (siRNAs) are a new class of promising therapeutic molecules that can be used for sequence-specific downregulation of disease-causing genes. However, endosomal entrapment of siRNA is a key hurdle for most delivery strategies, limiting the therapeutic effect. Here, we use live-cell microscopy and cytosolic galectin-9 as a sensor of membrane damage, to probe fundamental properties of endosomal escape of cholesterol-conjugated siRNA induced by endosome-disrupting compounds. We demonstrate efficient release of ligand-conjugated siRNA from vesicles damaged by small molecules, enhancing target knockdown up to ∼47-fold in tumor cells. Still, mismatch between siRNA-containing and drug-targeted endolysosomal compartments limits siRNA activity improvement. We also show widespread endosomal damage in macroscopic tumor spheroids after small molecule treatment, substantially improving siRNA delivery and knockdown throughout the spheroid. We believe the strategy to characterize endosomal escape presented here will be widely applicable, facilitating efforts to improve delivery of siRNA and other nucleic acid-based therapeutics.

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author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Nature Communications
volume
11
issue
1
article number
1809
publisher
Nature Publishing Group
external identifiers
  • scopus:85083546139
  • pmid:32286269
ISSN
2041-1723
DOI
10.1038/s41467-020-15300-1
language
English
LU publication?
yes
id
38750ad1-8a79-4d58-abd6-5d8d92f31f44
date added to LUP
2020-04-30 10:32:05
date last changed
2024-06-26 14:27:13
@article{38750ad1-8a79-4d58-abd6-5d8d92f31f44,
  abstract     = {{<p>Small interfering RNAs (siRNAs) are a new class of promising therapeutic molecules that can be used for sequence-specific downregulation of disease-causing genes. However, endosomal entrapment of siRNA is a key hurdle for most delivery strategies, limiting the therapeutic effect. Here, we use live-cell microscopy and cytosolic galectin-9 as a sensor of membrane damage, to probe fundamental properties of endosomal escape of cholesterol-conjugated siRNA induced by endosome-disrupting compounds. We demonstrate efficient release of ligand-conjugated siRNA from vesicles damaged by small molecules, enhancing target knockdown up to ∼47-fold in tumor cells. Still, mismatch between siRNA-containing and drug-targeted endolysosomal compartments limits siRNA activity improvement. We also show widespread endosomal damage in macroscopic tumor spheroids after small molecule treatment, substantially improving siRNA delivery and knockdown throughout the spheroid. We believe the strategy to characterize endosomal escape presented here will be widely applicable, facilitating efforts to improve delivery of siRNA and other nucleic acid-based therapeutics.</p>}},
  author       = {{Du Rietz, Hampus and Hedlund, Hampus and Wilhelmson, Sten and Nordenfelt, Pontus and Wittrup, Anders}},
  issn         = {{2041-1723}},
  language     = {{eng}},
  number       = {{1}},
  publisher    = {{Nature Publishing Group}},
  series       = {{Nature Communications}},
  title        = {{Imaging small molecule-induced endosomal escape of siRNA}},
  url          = {{http://dx.doi.org/10.1038/s41467-020-15300-1}},
  doi          = {{10.1038/s41467-020-15300-1}},
  volume       = {{11}},
  year         = {{2020}},
}