The use of oligonucleotides as therapeutic agents is of great interest, particularly when it comes to siRNA and antisense oligonucleotides. However, the difficulty of targeting the right organ and achieving efficient delivery to the cell remains a problem. There is much research activity directed at addressing these issues, employing solutions such as modification of the oligonucleotide backbone or the use of a delivery agent attached to the oligonucleotide.
In the case of the latter, various molecules including lipids, cationic lipids, cell-penetrating peptides and cell-targeting ligands have been reported to improve cell delivery by virtue of their hydrophobicity. This assists in their interaction with the hydrophobic environment of the cell membrane lipid bi-layer.
While these approaches have all been shown to enhance delivery, and in some cases even improve cell targeting, there are often associated challenges. Principally, even with successful delivery, there is still a need to release the oligonucleotide from the delivery agent once it has entered the cell to ensure efficient antisense activity.
One method of achieving this is the use of a cleavable disulphide bridge between the delivery agent and the oligonucleotide. However, when used in vivo there is the potential for toxic effects, since the delivery agents are frequently not recognized by the cells.
Cell Delivery Using Tocopherol
The use of vitamins as delivery agents has been shown to have potential in resolving this issue. Although vitamins such as tocopherol are not produced by the target cells, they are recognized by them, thus avoiding the issues of toxicity. They are also thought to be internalized by cells only after interaction with a binding protein, opening up the potential to target specific cell types using the technique.
Oligonucleotides modified with tocopherol via a disulphide-bridge are excellent candidates for the efficient delivery of factors such as therapeutic drugs directly into living cells. To make it easy to synthesize such custom oligonucleotides, Link Technologies has developed a range of tocopherol-based reagents. These simplify the incorporation of tocopherol into the 5´-end of an oligonucleotide and can be easily used with other modifications.
Modeling the Delivery and Release of Tocopherol-Modified Oligos
Although the direct incorporation of tocopherol into a therapeutic oligonucleotide should efficiently mediate its delivery into a cell, its hydrophobic nature might inhibit the final function of the oligonucleotide once inside the cell. To this end, we synthesized oligonucleotides conjugated to tocopherol via a cleavable disulphide-bridge.
After delivery to the cell, the tocopherol could be easily removed from the oligonucleotide by breaking the disulphide bridge, leaving the oligonucleotide free to carry out its therapeutic function. To demonstrate the feasibility of this approach, we constructed a cell delivery model system in which a hydrophobic cartridge takes the place of the cell membrane.
Oligonucleotides were synthesized using standard phosphoramidite chemistry, introducing a disulphide bridge linker followed by tocopherol incorporation at the 5´ end. The resulting crude oligonucleotide has the general structure tocopherol-S-S-oligo. After synthesis, the oligonucleotide was cleaved and deprotected, then immediately loaded onto a standard purification cartridge (Figure 1).
The purification method mimics what we would expect to happen in cell delivery, in that the modified oligonucleotide can be “delivered” to the cartridge and then the active molecule released via cleavage of the S-S bond, retaining the delivery agent (tocopherol) on the cartridge.
Using thiol-modified oligonucleotides conjugated to tocopherol as an example, we have generated data indicating that tocopherol successfully mediates attachment of the conjugate to the cartridge, and that the oligonucleotide can be released at a high level of purity following cleavage of the disulphide bridge.
This data indicates that the oligonucleotide can be easily released from the tocopherol delivery agent with sufficient efficiency and purity to accurately carry out its therapeutic function.