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Efficient delivery of triplex forming oligonucleotides to tumor cells by adenovirus-polylysine complexes
S W Ebbinghaus1, N Vigneswaran, C R Miller
1University of Alabama at Birmingham, USA.
Abstract:
Oligonucleotides (ODNs) show great promise in their ability to specifically inhibit single gene expression but must cross the cell membrane, escape the endosomal vesicle, and possibly traverse the nuclear membrane to arrive at their intracellular target molecules. In an attempt to improve the delivery of phosphodiester triplex forming ODNs to malignant cells, we have constructed adenovirus-polylysine (AdpL)-ODN complexes designed to take advantage of the receptor mediated endocytosis of adenoviruses to transfer the ODNs to the cell nucleus. Treatment of several different types of tumor cells in culture by AdpL-ODN complex resulted in superior uptake and persistence of the ODNs compared to both free ODN and cationic lipid-ODN complexes. Nuclear uptake peaks at 4 h and intact ODN persists in the nucleus with a half-life of 12 h. ODN concentrations of 20-70 microM are achieved at 24 h in all monolayer cell lines evaluated to date. ODNs are detected in 50-100% of the total cell population by immunohistochemistry with apparent uptake into vesicles and nuclear localization. Luciferase expression of a co-delivered reporter plasmid suggests that these ODNs are free in the nucleus. AdpL-ODN complexes will provide a valuable tool for delivering unmodified ODNs to the nucleus of malignant cells.
Insights
Adenovirus-polylysine (AdpL)-oligonucleotide (ODN) complexes enhance delivery of ODNs to malignant cells. This novel method improves ODN uptake and nuclear persistence for gene expression inhibition.
Area of Science:
- Molecular Biology
- Gene Therapy
- Nanotechnology
Background:
- Oligonucleotides (ODNs) are promising for gene expression inhibition but face delivery challenges.
- Efficient intracellular delivery is crucial for ODN therapeutic applications, especially to malignant cells.
- Current delivery methods like cationic lipids have limitations in achieving high intracellular concentrations and nuclear localization.
Purpose of the Study:
- To develop and evaluate an improved delivery system for phosphodiester triplex-forming ODNs to malignant cells.
- To utilize adenovirus-mediated endocytosis for enhanced ODN transport to the cell nucleus.
- To assess the efficacy of adenovirus-polylysine (AdpL)-ODN complexes in terms of cellular uptake, nuclear localization, and persistence.
Main Methods:
- Construction of AdpL-ODN complexes for targeted delivery.
- Treatment of various tumor cell lines in culture with AdpL-ODN complexes.
- Comparison of AdpL-ODN complexes with free ODN and cationic lipid-ODN complexes.
- Assessment of ODN uptake, nuclear localization, and persistence using immunohistochemistry and reporter gene assays.
Main Results:
- AdpL-ODN complexes demonstrated superior cellular uptake and ODN persistence compared to free ODN and lipid-ODN complexes.
- Nuclear uptake of ODNs peaked at 4 hours, with a half-life of 12 hours in the nucleus.
- High ODN concentrations (20-70 microM) were achieved within 24 hours in evaluated cell lines.
- Immunohistochemistry confirmed ODN presence in 50-100% of cells, with apparent vesicular and nuclear localization.
Conclusions:
- AdpL-ODN complexes represent a highly effective tool for delivering unmodified ODNs to the nucleus of malignant cells.
- The adenovirus-mediated delivery system overcomes key barriers in ODN intracellular transport.
- This approach holds significant potential for advancing gene therapy strategies targeting cancer and other diseases.
