Cholesterol conjugated oligonucleotide and LNA: a comparison of cellular and nuclear uptake by Hep2 cells enhanced by
Sárka Holasová1, Martin Mojzísek, Martin Buncek
1GENERI BIOTECH s.r.o., Hradec Králové, Czech Republic. sarka.holasova@generi-biotech.com
Abstract:
Antisense and antigene oligonucleotides (ONs) are attractive drugs for gene therapy, but major limiting factors for their routine use are inefficient cellular uptake and low accessibility to the target sites. Adding various lipophilic conjugates to the ON improves intracellular delivery as has been previously reported. We studied the cellular delivery of various ON modifications, as well as their cytosolic and nuclear distribution in mammalian Hep2-EGFP-NLS cell line. We compared uptake efficacy of ON and LNA, both conjugated with cholesterol at the 5' end. All ONs were 3' labeled with fluorescent Cy 5 dye. We made a comparison of the ONs uptake efficacy and the kinetics, both adding ONs to the culture medium, and using streptolysin-O (SL-O) permeabilization. The cellular uptake of each ON used in this study was visualized by fluorescent microscopy. We confirmed the results by FACS analysis. We determined the ratio between initial ON-chol concentration (0.4 microM) and the final amount in nucleus.SL-O can highly improve kinetics of ON delivery; not only into the cytoplasm but also to the nucleus, the presumed site of antigene ON action. The most effective nuclear uptake was observed when ON conjugated with cholesterol (ON-chol) and SL-O was used. Nuclear distribution of ON was reached within few minutes. In contrast, ON simply added to the medium reached cytoplasm only and the process of delivery took several hours.
Insights
Cholesterol-conjugated oligonucleotides (ONs) show enhanced cellular uptake and nuclear delivery. Streptolysin-O (SL-O) significantly improves delivery kinetics, enabling rapid nuclear access for gene therapy applications.
Area of Science:
- Molecular Biology
- Gene Therapy
- Drug Delivery
Background:
- Antisense and antigene oligonucleotides (ONs) are promising gene therapy agents.
- Inefficient cellular uptake and target site accessibility limit their clinical use.
- Lipophilic conjugates can enhance oligonucleotide intracellular delivery.
Purpose of the Study:
- To investigate the cellular delivery and distribution of modified oligonucleotides (ONs).
- To compare the uptake efficacy and kinetics of different ON modifications.
- To evaluate the role of cholesterol conjugation and streptolysin-O (SL-O) in ON delivery.
Main Methods:
- Utilized Hep2-EGFP-NLS cell line for mammalian cell studies.
- Conjugated cholesterol to 5' end of ONs and LNA; labeled 3' end with Cy 5.
- Compared ON uptake via culture medium addition versus SL-O permeabilization.
- Visualized cellular uptake via fluorescent microscopy and confirmed with FACS analysis.
Main Results:
- SL-O significantly improved the kinetics of ON delivery into both cytoplasm and nucleus.
- Cholesterol-conjugated ONs (ON-chol) combined with SL-O demonstrated the most effective nuclear uptake.
- Nuclear distribution of ONs was achieved within minutes using SL-O, compared to hours for medium addition.
- Standard medium addition resulted in cytoplasmic delivery only.
Conclusions:
- SL-O is a highly effective method for enhancing oligonucleotide delivery kinetics.
- Cholesterol conjugation of ONs, particularly with SL-O, facilitates rapid and efficient nuclear delivery.
- These findings suggest improved strategies for utilizing ONs in gene therapy by overcoming delivery barriers.
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