Intracellular Distribution and Nuclear Activity of Antisense Oligonucleotides After Unassisted Uptake in Myoblasts and Differentiated MyotubesIn Vitro

Abstract

Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of AONs with highest in vitro efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood. Here, we report on the unassisted uptake of naked AONs, so-called gymnosis, in muscle cells in culture. We found that gymnosis works similarly well for proliferating myoblasts as for terminally differentiated myotubes. Cell biological analyses combined with microscopy imaging showed that a phosphorothioate backbone promotes efficient gymnosis, that uptake is clathrin mediated and mainly results in endosomal-lysosomal accumulation. Nuclear localization occurred at a low level, but the gymnotically delivered AONs effectively modulated the expression of their nuclear RNA targets. Chloroquine treatment after gymnotic delivery helped increase nuclear AON levels. In sum, we demonstrate that gymnosis is feasible in proliferating and non-proliferating muscle cells and we confirm the relevance of AON chemistry for uptake and intracellular trafficking with this method, which provides a useful means for bio-activity screening of AONs in vitro.

Nuclear and Cytoplasmatic Quantification of Unconjugated, Label-Free Locked Nucleic Acid Oligonucleotides

“…At present, most studies on intracellular presence and trafficking rely on covalent attachment of a fluorophore onto the oligonucleotide [38][39][40][41][42][43]. These studies involving fluorescently labeled oligonucleotides are beneficial to understanding the distribution, and some constructs mimic very well the situation of unconjugated AON [44].…”

The nuclear concentration required for antisense oligonucleotide activity in myotonic dystrophy cells

“…Neither for R9-nor for gymnotically delivered ASOs did we observe such a localization. This was surprising, as in a previous study we had described nuclear localization and efficacy of gymnotically delivered ASOs, but this discrepancy can be explained by the much longer incubation times used earlier, as well as intrinsic differences between murine and human myoblasts (44). Fluorimetry on cell lysates further showed that the uptake of ASOs was more than 10-fold higher in cells that were incubated with PF14 polyplexes compared with R9 polyplexes or naked ASOs, which is in agreement with a previous report (32).…”

“…Thus, areas that showed highly fluorescent spots from other focal planes than the nucleus were distinguished from those nuclei that had a homogeneous fluorescence distribution. For each of the 3 independent uptake experiments, between 57 and 103 nuclei were measured (untreated control [38][39][40][41][42][43][44][45][46][47][48]. A 1-way ANOVA was performed to test whether there was a treatment effect.…”

A critical analysis of methods used to investigate the cellular uptake and subcellular localization of RNA therapeutics

“…However, immunofluorescence must be performed on fixed and permeabilized cells in order to deliver the antibodies to the interior of the cell. Oligonucleotides and other biomolecules have been shown to redistribute throughout the cell as a result of fixation, leading to false-positive artifacts ( 33 , 51 , 86 , 108 , 109 ). Finally, even with careful colocalization studies, it can be difficult to deconvolute cytosolic versus endosomal material in a definitive manner.…”

“…After treatment with the RNA therapeutic, cells were fixed and permeabilized prior to staining with antibodies against specific endocytic markers. As several studies have shown ( 33 , 51 , 86 , 108 , 109 ), fixation can allow for redistribution of oligonucleotides and other biomolecules during subsequent incubation steps. When investigating subcellular localization, it is inherently problematic to interpret results from fixed and permeabilized cells.…”