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

Antisense oligonucleotides (ASOs) are a promising class of therapeutics that are starting to emerge in the clinic. Determination of intracellular concentrations required for biologic effects and identification of effective delivery vehicles are crucial for understanding the mode of action and required dosing. Here, we investigated which nuclear oligonucleotide concentration is needed for a therapeutic effect for a triplet repeat-targeting ASO in a muscle cell model of myotonic dystrophy type 1 (DM1). For cellular delivery, ASOs were complexed into nanoparticles using the cationic cell-penetrating peptides nona-arginine and PepFect14 (PF14). Although both peptides facilitated uptake, only PF14 led to a dose-dependent correction of disease-typical abnormal splicing. In line with this observation, time-lapse confocal microscopy demonstrated that only PF14 mediated translocation of the ASOs to the nucleus, which is the main site of action. Through fluorescence lifetime imaging, we could distinguish intact oligonucleotide from free fluorophore, showing that PF14 also shielded the ASOs from degradation. Finally, we employed a combination of live-cell fluorescence correlation spectroscopy and immunofluorescence microscopy and demonstrated that intranuclear blocking-type oligonucleotide concentrations in the upper nanomolar range were required to dissolve nuclear muscleblind-like protein 1 foci, a hallmark of DM1. Our findings have important implications for the clinical use of ASOs in DM1 and provide a basis for further research on other types of ASOs.-Van der Bent, M. L., Paulino da Silva Filho, O., Willemse, M., Hällbrink, M., Wansink, D. G., Brock, R. The nuclear concentration required for antisense oligonucleotide activity in myotonic dystrophy cells.