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A two step model aimed at delivering antisense oligonucleotides in targeted cells
J Toth1, I Boszormenyi, Z S Majer
1CNRS UMR 1582, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif cedex, France.
Abstract:
To be efficient in vivo antisense oligonucleotides must reach the targeted cells and then cross the cellular membrane. We propose a two step system where the oligonucleotide is first electrostatically bound to a peptide coupled to a ligand of a cellular receptor. A complex is formed which allows the oligonucleotide to be bound to the membrane of the targeted cells. These oligonucleotides are then delivered inside the cells by the subsequent use of a transfection agent. As a reductionist model of peptide coupled to a ligand we have used a lipopeptide and characterized by a filter elution assay the stoichiometry between the peptide and the oligonucleotide in the complexes. Using HeLa cultured cells we have shown that addition of these complexes to the cells triggers the oligonucleotide binding to the cell membrane. The subsequent addition of dendrimers allows these antisense oligonucleotides to inhibit a reporter gene inside the cells.
Insights
This study presents a novel two-step system for delivering antisense oligonucleotides into cells. The system uses a peptide-ligand complex to bind oligonucleotides to cell membranes, followed by dendrimers for intracellular delivery, enabling gene inhibition.
Area of Science:
- Molecular Biology
- Biotechnology
- Drug Delivery
Background:
- Antisense oligonucleotides (ASOs) require efficient cellular uptake for in vivo efficacy.
- Current delivery methods face challenges in targeting specific cells and crossing the cell membrane.
Purpose of the Study:
- To develop and validate a two-step system for enhanced cellular delivery of antisense oligonucleotides.
- To demonstrate the ability of this system to facilitate gene inhibition within targeted cells.
Main Methods:
- Formation of electrostatic complexes between oligonucleotides, a lipopeptide (peptide-ligand model), and a cellular receptor ligand.
- Characterization of peptide-oligonucleotide stoichiometry using a filter elution assay.
- In vitro studies using HeLa cells to assess membrane binding and subsequent intracellular delivery via transfection agents (dendrimers).
- Reporter gene assays to evaluate the functional inhibition mediated by delivered ASOs.
Main Results:
- Successfully formed complexes enabling electrostatic binding of ASOs to cell membranes.
- Demonstrated that the lipopeptide-oligonucleotide complexes bind to HeLa cell membranes.
- Showed that subsequent addition of dendrimers facilitates intracellular delivery of ASOs.
- Confirmed that delivered ASOs can inhibit reporter gene expression within cells.
Conclusions:
- The proposed two-step system effectively targets and delivers antisense oligonucleotides into cells.
- This approach holds promise for improving the in vivo efficiency of oligonucleotide-based therapeutics.
- Further development could lead to advanced gene silencing strategies.