The primary goal of this study was to track PS-ASO and GalNAc-PS-ASO uptake in two cell cultures as the first step to understand the observations from the clinical studies. The multimodal imaging setup of CARS and 2PF modalities in conjunction with the image analysis pipeline made it uniquely possible to address these challenges. We report here the time-dependent uptake, internalization, and localization differences between GalNAc-PS-ASOs and PS-ASOs in liver cells. We believe our findings will help us form the basis for further investigations with more complex cellular co-cultures and with tissue and animal models.
Antisense oligonucleotides (ASOs) are single stranded negatively charged molecules which downregulate the translation of specific target messenger RNA (mRNA). Chemically modified ASOs with phosphorothioate (PS) linkages have been extensively studied as research tools and as clinical therapeutics and nine oligonucleotide-based drugs have been approved by regulatory agencies. While several cell surface proteins that bind PS-ASOs and mediate their cellular uptake have been identified, the mechanisms leading to productive internalization of PS-ASOs are not well understood. We demonstrate the potential of hyperspectral CARS imaging to detect the intracellular presence of ASOs in a label-free manner.
Antisense oligonucleotides (ASOs), a novel paradigm in modern therapeutics, modulate cellular gene expression by binding to complementary RNA sequences. Triantennary N-acetyl galactosamine (GN)-conjugated ASOs show greatly improved potency via Asialoglycoprotein receptor (ASGR)-mediated uptake in hepatocytes. Here, we compare the uptake kinetics and subsequent distribution of untargeted ASOs to that of GN-ASOs in mouse macrophages and hepatocytes using simultaneous coherent anti-Stokes Raman scattering (CARS) and two-photon excited fluorescence imaging. While the CARS modality captured the changing lipid distributions and overall morphology of the cell, two-photon fluorescence imaging measured the uptake and the subsequent distribution of the fluorescently labeled (Alexa-488) ASOs inside the cells.
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