Localization of unlabeled bepirovirsen antisense oligonucleotide in murine tissues using in situ hybridization and CARS imaging.
antisense oligonucleotides
coherent anti-Stokes Raman scattering
in situ hybridization
label-free imaging
miRNAscope
multiphoton imaging
pharmacokinetics
Journal
RNA (New York, N.Y.)
ISSN: 1469-9001
Titre abrégé: RNA
Pays: United States
ID NLM: 9509184
Informations de publication
Date de publication:
10 2023
10 2023
Historique:
received:
27
04
2023
accepted:
29
06
2023
medline:
20
9
2023
pubmed:
18
7
2023
entrez:
17
7
2023
Statut:
ppublish
Résumé
Current methods for detecting unlabeled antisense oligonucleotide (ASO) drugs rely on immunohistochemistry (IHC) and/or conjugated molecules, which lack sufficient sensitivity, specificity, and resolution to fully investigate their biodistribution. Our aim was to demonstrate the qualitative and quantitative distribution of unlabeled bepirovirsen, a clinical stage ASO, in livers and kidneys of dosed mice using novel staining and imaging technologies at subcellular resolution. ASOs were detected in formalin-fixed paraffin-embedded (FFPE) and frozen tissues using an automated chromogenic in situ hybridization (ISH) assay: miRNAscope. This was then combined with immunohistochemical detection of cell lineage markers. ASO distribution in hepatocytes versus nonparenchymal cell lineages was quantified using HALO AI image analysis. To complement this, hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) imaging microscopy was used to specifically detect the unique cellular Raman spectral signatures following ASO treatment. Bepirovirsen was localized primarily in nonparenchymal liver cells and proximal renal tubules. Codetection of ASO with distinct cell lineage markers of liver and kidney populations aided target cell identity facilitating quantification. Positive liver signal was quantified using HALO AI, with 12.9% of the ASO localized to the hepatocytes and 87.1% in nonparenchymal cells. HS-CARS imaging specifically detected ASO fingerprints based on the unique vibrational signatures following unlabeled ASO treatment in a totally nonperturbative manner at subcellular resolution. Together, these novel detection and imaging modalities represent a significant increase in our ability to detect unlabeled ASOs in tissues, demonstrating improved levels of specificity and resolution. These methods help us understand their underlying mechanisms of action and ultimately improve the therapeutic potential of these important drugs for treating globally significant human diseases.
Identifiants
pubmed: 37460153
pii: rna.079699.123
doi: 10.1261/rna.079699.123
pmc: PMC10578491
doi:
Substances chimiques
Oligonucleotides, Antisense
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1575-1590Informations de copyright
© 2023 Spencer-Dene et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
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