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• minimal toxicity: thus far, most trials report few or no side effects. This is likely due to the highly specific nature of oligo duplexing, and the fact that they are ‘natural’ biomolecules;
• the requirement for only low levels of the oligo to be present inside the cell, as target mRNA is, itself, usually present only in nanomolar (nM) concentrations;
• the ability to manufacture oligos of specified nucleotide sequence is relatively straightforward using automated synthesizers.
However, native antisense oligonucleotides also suffer from a number of disadvantages, which include:
• sensitivity to nucleases;
• very low serum half-lives;
Linkage name Substituent (R)
Figure 11.14. Major types of modification potentially made to an oligo’s phosphodiester linkage in order to increase its stability or enhance some other functional characteristic
• poor rate of cellular uptake;
• orally inactive.
Some progress has been made in overcoming such difficulties, and continued progress in the area is expected to render the next generation of oligos more therapeutically effective.
Native oligonucleotides display a 3'-5' phosphodiester linkage in their backbone (Figure 11.14). These are sensitive to a range of nucleases naturally present in most extracellular fluids and intracellular compartments. The half-life of native oligonucleotides in serum is only ca. 15 min, and oligoribonucleotides are less stable than oligodeoxynucleotides. Selective modification of the native phosphodiester bond can render the product resistant to nuclease degradation.
Modification usually entails replacement of one of the free (non-bridging) oxygens of the phosphodiester linkage with an alternative atom or chemical group (Figure 11.14). Most commonly, the oxygen has been replaced with a sulphur atom and the resultant phosphoro-thioates display greatest clinical promise. Phosphorothioate-based oligos (‘S’-oligos), display increased resistance to nuclease attack, while remaining water-soluble. They are also easy to
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synthesize chemically and display a biological half-life of up to 24 h. Most antisense oligos currently being assessed in clinical trials are S-oligos.
Delivery and cellular uptake of oligonucleotides
Oligo administration during many clinical trials entails direct i.v. infusion, often over a course of several hours. Although relatively stable in serum, the commonly employed phosphorothioate oligos (and indeed most other oligo types) encounter several barriers to reaching their final destinations. They bind various serum proteins, including serum albumin, as well as a range of heparin-binding and other proteins, which commonly occur on many cell surfaces. Targeting of naked oligos to specific cell types is therefore not possible. Following administration, these oligos tend to be distributed to many tissues, with the highest proportion accumulating in the liver and kidney.
The precise mechanism(s) by which oligos enter cells are not fully understood. Most are charged molecules, sometimes displaying a molecular mass of up to 10-12 kDa. Receptor-mediated endocytosis appears to be the most common mechanism by which charged oligos, such as phosphorothioates, enter most cells. One putative phosphorothioate receptor appears to consist of an 80 kDa surface protein, associated with a smaller 34 kDa membrane protein. However, this in itself seems to be an inefficient process, with only a small proportion of the administered drug eventually being transferred across the plasma membrane.
Uncharged oligos appear to enter the cell by passive diffusion, as well as possibly by endocytosis. However, elimination of the charges renders the resultant oligos relatively hydrophobic, thus generating additional difficulties with their synthesis and delivery.
Attempts to increase delivery of oligos into the cell centre mainly on the use of suitable carrier systems. Liposomes, as well as polymeric carriers (e.g. polylysine-based carriers), are gaining most attention in this regard. Details of such carriers have already been discussed earlier in this chapter.
An alternative system, which effectively results in the introduction of antisense oligonucleotides into the cell, entails the application of gene therapy. In this case, a gene which, when transcribed, yields (antisense) mRNA of appropriate nucleotide sequence, is introduced into the cell by a retroviral or other appropriate vector. This approach, as applied to the treatment of cancer and AIDS, is being appraised in a number of trials.
Oligos, including modified oligos, appear to be ultimately metabolized within the cell by the action of nucleases, particularly 3'-exonucleases. Breakdown metabolic products are then mainly excreted via the urinary route.