Localization of unlabeled bepirovirsen antisense oligonucleotide in murine tissues using in situ hybridization and CARS imaging

Abstract

Current methods for detecting unlabelled ASOs rely on immunohistochemistry and/or conjugated molecules, which lack sufficient sensitivity, specificity and resolution to fully investigate their bio-distribution. Our aim was to demonstrate the qualitative and quantitative distribution of unlabelled bepirovirsen, a clinical stage ASO, in livers and kidneys of dosed mice using novel staining and imaging technologies at sub-cellular resolution. ASOs were detected in FFPE and frozen tissues using an automated chromogenic in situ hybridization assay: miRNAscope. This was then combined with immunohistochemical detection of cell lineage markers. ASO distribution in hepatocytes versus non-parenchymal cell lineages, was quantified using HALO AI image analysis. To complement this, Coherent anti-Stokes Raman Scattering (CARS) imaging microscopy was used to specifically detect the unique cellular Raman spectral signatures following ASO treatment. Bepirovirsen was localized primarily in non-parenchymal liver cells and proximal renal tubules. Co-detection 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 non-parenchymal cells. CARS imaging specifically detected the unique vibrational signatures following unlabelled ASO treatment in a totally non-perturbative manner at subcellular resolution. Together, these novel detection and imaging modalities represent a significant increase in our ability to detect unlabelled 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.

Innovative Approaches for Drug Discovery: Quantifying Drug Distribution and Response with Raman Imaging

“…In renal tubules, Sperati et al identified the spectroscopic signature of intratubular triamterene crystals, a diuretic that is linked to kidney stone formation . Spencer-Dene et al combined CARS imaging with an miRNAscope in situ hybridization (ISH) assay to monitor the distribution of bepirovirsen in murine kidney and liver tissues . While a few studies have explored direct imaging of drugs in internal organ tissue, the overwhelming majority has focused on the skin.…”

“…95 Spencer-Dene et al combined CARS imaging with an miRNAscope in situ hybridization (ISH) assay to monitor the distribution of bepirovirsen in murine kidney and liver tissues. 96 While a few studies have explored direct imaging of drugs in internal organ tissue, the overwhelming majority has focused on the skin. This is likely due to the accessibility and integrity of skin tissue samples and their ease of preparation for imaging.…”