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Abstract
Locked nucleic acid based antisense oligonucleotides (LNA-ASOs) can reach their intracellular RNA targets without delivery modules. Functional cellular uptake involves vesicular accumulation followed by translocation to the cytosol and nucleus. However, it is yet unknown how many LNA-ASO molecules need to be delivered to achieve target knock down. Here we show by quantitative fluorescence imaging combined with LNA-ASO microinjection into the cytosol or unassisted uptake that ∼105 molecules produce >50% knock down of their targets, indicating that a substantial amount of LNA-ASO escapes from endosomes. Microinjected LNA-ASOs redistributed within minutes from the cytosol to the nucleus and remained bound to nuclear components. Together with the fact that RNA levels for a given target are several orders of magnitude lower than the amounts of LNA-ASO, our data indicate that only a minor fraction is available for RNase H1 mediated reduction of target RNA. When non-specific binding sites were blocked by co-administration of non-related LNA-ASOs, the amount of target LNA-ASO required was reduced by an order of magnitude. Therefore, dynamic processes within the nucleus appear to influence the distribution and activity of LNA-ASOs and may represent important parameters for improving their efficacy and potency.
Highlights
Antisense technologies have experienced significant interest in academia and industry both as research tools and therapeutic agents
We analyzed the efficacy of target knock down in the injected cells either on the RNA or on the protein levels via quantitative fluorescence imaging of single cells
Intracellular concentrations obtained from confocal imaging in photon counting mode were validated by fluorescence correlation spectroscopy (FCS), a technique that measures intensity fluctuations within the focal volume depending on the fluorophore concentration (Supplementary Figure S2) [41]
Summary
Antisense technologies have experienced significant interest in academia and industry both as research tools and therapeutic agents. Oligonucleotide based modalities have shown great promise because of their superior target selectivity and potency against otherwise undruggable RNA targets They can suppress gene expression, modulate mRNA splicing or target non-coding RNAs (ncRNAs) involved in transcriptional and epigenetic regulation [1,2,3,4,5]. To reach their intracellular sites of action, oligonucleotides need to overcome cellular membrane barriers such as the plasma membrane and/or the limiting membrane of endosomes [6]. Gymnosis alleviates the need for transfection reagents and IC50 values are typically found in the micromolar to submicromolar range [10,11,12,13]
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