In Vitro/In Vivo Translation, BioImaging, GSK, Stevenage SG1 2NY, United Kingdom.
GSK Center for Optical Molecular Imaging, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
RNA. 2023 Oct;29(10):1575-1590. doi: 10.1261/rna.079699.123. Epub 2023 Jul 17.
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.
当前检测未标记反义寡核苷酸（ASO）药物的方法依赖于免疫组织化学（IHC）和/或缀合分子，但这些方法缺乏足够的灵敏度、特异性和分辨率，无法充分研究其生物分布。我们的目的是使用新型染色和成像技术在亚细胞分辨率下，证明临床阶段 ASO 药物 bepirovirsen 在给药小鼠的肝脏和肾脏中的定性和定量分布。使用自动化显色原位杂交（ISH）检测试剂盒 miRNAscope 在福尔马林固定石蜡包埋（FFPE）和冷冻组织中检测 ASOs：这与细胞谱系标志物的免疫组织化学检测相结合。使用 HALO AI 图像分析对肝细胞与非实质细胞谱系中的 ASO 分布进行定量。为了补充这一点，使用高光谱相干反斯托克斯拉曼散射（HS-CARS）成像显微镜来特异性检测 ASO 处理后独特的细胞拉曼光谱特征。Bepirovirsen 主要定位于非实质肝细胞和近端肾小管。用肝脏和肾脏细胞群的不同细胞谱系标志物共检测 ASO 有助于确定靶细胞身份并促进定量。使用 HALO AI 定量阳性肝脏信号，12.9%的 ASO 定位于肝细胞，87.1%定位于非实质细胞。HS-CARS 成像以非侵入性方式特异性检测未标记 ASO 处理后基于独特振动特征的 ASO 指纹，以亚细胞分辨率实现。这些新型检测和成像方法的结合代表了我们在组织中检测未标记 ASO 的能力有了显著提高，表现出更高的特异性和分辨率。这些方法有助于我们了解它们的作用机制，并最终提高这些治疗全球重大人类疾病的重要药物的治疗潜力。
