Related Experiment Video
Updated: Oct 1, 2025
In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge
Published on: June 15, 2018
Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides
Jeremy P Bost1, Miina Ojansivu2, Michael J Munson3
1Department of Laboratory Medicine, Karolinska Institutet, Huddinge, 14157, Sweden. jeremy.bost@ki.se.
Abstract:
The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.
Insights
Novel endosomolytic compounds enhance splice-switching oligonucleotide (SSO) delivery by promoting escape from endosomes. These compounds facilitate SSO translocation into the cytosol, improving therapeutic potential.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Delivery
Background:
- Oligonucleotides (ONs) face delivery challenges due to endosomal entrapment, limiting their therapeutic and research applications.
- Splice-switching ONs (SSOs) require endosomolytic agents for enhanced cytosolic and nuclear access.
- Current methods lack high resolution to link SSO trafficking with activity.
Purpose of the Study:
- To characterize novel endosomolytic compounds for improved SSO delivery.
- To develop high-resolution methods correlating SSO trafficking with functional activity.
- To evaluate the impact of endosomolytic compounds on SSO translocation and endosomal escape.
Main Methods:
- Utilized high spatiotemporal resolution microscopy and functional assays.
- Visualized SSO trafficking and endosomal membrane rupture.
- Quantified SSO functional activity at the protein level.
Main Results:
- Confirmed SSO leakage into the cytosol occurs with endosomal engorgement (LAMP1-positive).
- Demonstrated new compounds disrupt SSO trafficking to LAMP1-positive compartments.
- Showed compounds induce endosomal membrane rupture and concurrent ON escape.
Conclusions:
- Novel endosomolytic compounds effectively promote SSO escape from endosomes into the cytosol.
- These compounds represent potent, rapid transfection reagents for antisense ONs.
- The developed system enables detailed correlation of SSO trafficking and activity.
More Related Videos
09:04Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids
Published on: September 21, 2017
07:47Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System
Published on: October 29, 2019
Related Concept Videos
02:30siRNA - Small Interfering RNAs
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the...
02:15Experimental RNAi
01:23RNA Interference
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...