Efficient cationic lipid-mediated delivery of antisense oligonucleotides into eukaryotic cells: down-regulation of
1Department of Membrane Cell Biology, University of Groningen, Faculty of Medical Sciences, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Abstract:
Oligonucleotides (ODNs) can be employed as effective gene-specific regulators. However, before ODNs can reach their targets, several physical barriers have to be overcome, as although ODNs may pass cell membranes, most become sequestered in endocytic compartments. Accordingly, sophisticated strategies are required for efficient delivery. Here we have employed a pyridinium-based synthetic amphiphile, called SAINT-2, which carries ODNs into cells in a highly efficient, essentially non-toxic and serum-insensitive manner. Intracellular delivery was examined by monitoring the trafficking of fluorescent ODNs and lipid, and by measuring the effect of specific antisense ODNs on target mRNA and protein levels of the receptor for the neuropeptide corticotropin-releasing factor (CRF-R), expressed in Chinese hamster ovary cells. ODN delivery is independent of lipoplex size, and fluorescently tagged ODNs readily acquire access to the nucleus, whereas the carrier itself remains sequestered in the endosomal-lysosomal pathway. While the release is independent of the presence of serum, it is not observed when serum proteins gain access within the lipoplex, and which likely stabilizes the lipoplex membrane. We propose that the amphiphile-dependent aggregate structure governs complex dissociation, and hence, the biological efficiency of ODNS: We demonstrate an essentially non-toxic and effective antisense-specific down-regulation of the CRF-R, both at the mRNA and protein level.
Insights
This study introduces SAINT-2, a synthetic amphiphile for efficient and non-toxic delivery of oligonucleotides (ODNs) into cells. SAINT-2 facilitates gene regulation by overcoming cellular barriers, enabling effective antisense therapy.
Area of Science:
- Molecular Biology
- Biochemistry
- Drug Delivery Systems
Background:
- Oligonucleotides (ODNs) show promise as gene-specific regulators but face significant delivery challenges due to endosomal sequestration.
- Efficient intracellular delivery is crucial for ODN efficacy, necessitating advanced strategies to overcome physical barriers.
Purpose of the Study:
- To evaluate the efficacy and safety of a novel pyridinium-based synthetic amphiphile, SAINT-2, for delivering ODNs into cells.
- To investigate the intracellular trafficking and mechanism of ODN delivery mediated by SAINT-2.
- To demonstrate the functional application of SAINT-2-mediated ODN delivery in gene silencing.
Main Methods:
- Utilized fluorescently tagged ODNs and lipid (SAINT-2) to track intracellular delivery and trafficking.
- Assessed the impact of ODN delivery on target mRNA and protein levels using antisense ODNs against the corticotropin-releasing factor receptor (CRF-R).
- Examined the influence of lipoplex size and serum presence on ODN delivery efficiency and release.
Main Results:
- SAINT-2 demonstrated highly efficient, non-toxic, and serum-insensitive delivery of ODNs into cells.
- Fluorescently tagged ODNs accessed the nucleus, while the SAINT-2 carrier remained in the endosomal-lysosomal pathway.
- Delivery efficiency was independent of lipoplex size, and serum proteins could inhibit release by stabilizing the lipoplex membrane.
- Successfully achieved antisense-specific down-regulation of CRF-R at both mRNA and protein levels.
Conclusions:
- SAINT-2 is a potent and safe synthetic amphiphile for intracellular ODN delivery, overcoming common cellular barriers.
- The amphiphile's structure and its interaction with cellular components are critical for efficient complex dissociation and biological activity.
- This strategy holds significant potential for developing effective antisense-based gene therapies.
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