Localization of Unlabeled Bepirovirsen Antisense Oligonucleotide in Murine Tissues Using in Situ Hybridization and CARS Imaging

Overview

Journal RNA

Specialty Molecular Biology

Date 2023 Jul 17

PMID 37460153

Authors

Bradley Spencer-Dene

Prabuddha Mukherjee

Aneesh Alex

Kajari Bera

Wei-Ju Tseng

Jindou Shi

Eric J Chaney

Darold R Spillman Jr

Marina Marjanovic

Elena Miranda

Stephen A Boppart

Steve R Hood

Affiliations

Soon will be listed here.

Abstract

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.

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References

Han K, Theodore D, McMullen G, Swayze E, McCaleb M, Billioud G . Preclinical and Phase 1 Assessment of Antisense Oligonucleotide Bepirovirsen in Hepatitis B Virus-Transgenic Mice and Healthy Human Volunteers: Support for Clinical Dose Selection and Evaluation of Safety, Tolerability, and Pharmacokinetics of.... Clin Pharmacol Drug Dev. 2022; 11(10):1191-1202. DOI: 10.1002/cpdd.1154. View

Zhang C, Boppart S . Dynamic Signatures of Lipid Droplets as New Markers to Quantify Cellular Metabolic Changes. Anal Chem. 2020; 92(24):15943-15952. DOI: 10.1021/acs.analchem.0c03366. View

Billioud G, Kruse R, Carrillo M, Whitten-Bauer C, Gao D, Kim A . In vivo reduction of hepatitis B virus antigenemia and viremia by antisense oligonucleotides. J Hepatol. 2015; 64(4):781-9. DOI: 10.1016/j.jhep.2015.11.032. View

Chang Y, Yang C, Chen Y, Yang C, Chou Y, Chou H . A siRNA targets and inhibits a broad range of SARS-CoV-2 infections including Delta variant. EMBO Mol Med. 2022; 14(4):e15298. PMC: 8988202. DOI: 10.15252/emmm.202115298. View

Richter M, Deligiannis I, Yin K, Danese A, Lleshi E, Coupland P . Single-nucleus RNA-seq2 reveals functional crosstalk between liver zonation and ploidy. Nat Commun. 2021; 12(1):4264. PMC: 8275628. DOI: 10.1038/s41467-021-24543-5. View

Huser T, Chan J . Raman spectroscopy for physiological investigations of tissues and cells. Adv Drug Deliv Rev. 2015; 89:57-70. DOI: 10.1016/j.addr.2015.06.011. View

Acs B, Pelekanou V, Bai Y, Martinez-Morilla S, Toki M, Leung S . Ki67 reproducibility using digital image analysis: an inter-platform and inter-operator study. Lab Invest. 2018; 99(1):107-117. DOI: 10.1038/s41374-018-0123-7. View

Goebl N, Berridge B, Wroblewski V, Brown-Augsburger P . Development of a sensitive and specific in situ hybridization technique for the cellular localization of antisense oligodeoxynucleotide drugs in tissue sections. Toxicol Pathol. 2007; 35(4):541-8. DOI: 10.1080/01926230701338958. View

Geary R, Norris D, Yu R, Bennett C . Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv Drug Deliv Rev. 2015; 87:46-51. DOI: 10.1016/j.addr.2015.01.008. View

10.

Gallo Cantafio M, Nielsen B, Mignogna C, Arbitrio M, Botta C, Frandsen N . Pharmacokinetics and Pharmacodynamics of a 13-mer LNA-inhibitor-miR-221 in Mice and Non-human Primates. Mol Ther Nucleic Acids. 2016; 5(6). PMC: 5022129. DOI: 10.1038/mtna.2016.36. View

11.

Butler H, Ashton L, Bird B, Cinque G, Curtis K, Dorney J . Using Raman spectroscopy to characterize biological materials. Nat Protoc. 2016; 11(4):664-87. DOI: 10.1038/nprot.2016.036. View

12.

Juliano R, Ming X, Nakagawa O . Cellular uptake and intracellular trafficking of antisense and siRNA oligonucleotides. Bioconjug Chem. 2011; 23(2):147-57. PMC: 3288458. DOI: 10.1021/bc200377d. View

13.

Deprey K, Batistatou N, Debets M, Godfrey J, VanderWall K, Miles R . Quantitative Measurement of Cytosolic and Nuclear Penetration of Oligonucleotide Therapeutics. ACS Chem Biol. 2022; 17(2):348-360. PMC: 9252293. DOI: 10.1021/acschembio.1c00830. View

14.

Prakash T, Graham M, Yu J, Carty R, Low A, Chappell A . Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res. 2014; 42(13):8796-807. PMC: 4117763. DOI: 10.1093/nar/gku531. View

15.

Geary R . Antisense oligonucleotide pharmacokinetics and metabolism. Expert Opin Drug Metab Toxicol. 2009; 5(4):381-91. DOI: 10.1517/17425250902877680. View

16.

He C, Migawa M, Chen K, Weston T, Tanowitz M, Song W . High-resolution visualization and quantification of nucleic acid-based therapeutics in cells and tissues using Nanoscale secondary ion mass spectrometry (NanoSIMS). Nucleic Acids Res. 2020; 49(1):1-14. PMC: 7797060. DOI: 10.1093/nar/gkaa1112. View

17.

Vervaeke P, Borgos S, Sanders N, Combes F . Regulatory guidelines and preclinical tools to study the biodistribution of RNA therapeutics. Adv Drug Deliv Rev. 2022; 184:114236. PMC: 8957368. DOI: 10.1016/j.addr.2022.114236. View

18.

Butler M, Crooke R, Graham M, Lemonidis K, Lougheed M, Murray S . Phosphorothioate oligodeoxynucleotides distribute similarly in class A scavenger receptor knockout and wild-type mice. J Pharmacol Exp Ther. 2000; 292(2):489-96. View

19.

Holzer T, Hanson J, Wray E, Bailey J, Kennedy K, Finnegan P . Cross-Platform Comparison of Computer-assisted Image Analysis Quantification of In Situ mRNA Hybridization in Investigative Pathology. Appl Immunohistochem Mol Morphol. 2017; 27(1):15-26. DOI: 10.1097/PAI.0000000000000542. View

20.

Ait Benichou S, Jauvin D, De Serres-Berard T, Pierre M, Ling K, Bennett C . Antisense oligonucleotides as a potential treatment for brain deficits observed in myotonic dystrophy type 1. Gene Ther. 2022; 29(12):698-709. PMC: 9750879. DOI: 10.1038/s41434-022-00316-7. View