Biomedical Engineering Reference
In-Depth Information
Attempts to increase delivery of oligos into the cell mainly centre 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 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 modifi ed 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.
Even phosphorothioate oligos display serum and tissue half-lives of less than a day. As a con-
sequence, continuous or frequent i.v. infusions are required for product administration. Some
progress has been reported in the development of second-generation phosphorothioate oligos with
improved pharmacokinetic characteristics. The most promising development entails the modifi ca-
tion of the ribose sugar found in the repeat nucleotide structure. Attachment (at the 2´ position) of
methyl (!CH
3
) or methoxy ethyl (!CH
2
CH
2
OCH
3
) groups increases product stability, as well as
product potency (by enhancing binding affi nity for RNA). However, these changes also abrogate
the product's ability to activate RNaseH, a primary mechanism of inducing its antisense effect.
This, in turn, may be overcome by the more recent development of chimaeric phosphorothioate
oligos, in which 2´-modifi ed sugar nucleotides are placed only at the ends of the molecule, leaving
a nuclease-compatible gap in the middle.
14.8.1 Manufacture of oligos
In contrast to the biopharmaceuticals discussed thus far (recombinant proteins and gene therapy
products), antisense oligonucleotides are manufactured by direct chemical synthesis. Organic syn-
thetic pathways have been developed, optimized and commercialized for some time, as oligonu-
cleotides are widely used reagents in molecular biology. They are required as primers, probes and
for the purposes of site-directed mutagenesis.
The nucleotides required (themselves either modifi ed or unmodifi ed as desired) are fi rst reacted
with a protecting chemical group. Each protected nucleotide is then coupled in turn to the growing
end of the nucleotide chain, itself attached to a solid phase. After coupling, the original protecting
group is removed and, when chain synthesis is complete, the bond anchoring the chemical to the
solid phase is hydrolysed, releasing the free oligo. This may then be purifi ed by HPLC. The most
common synthetic method used is known as the phosphoramidite method, which uses a dimeth-
oxytrityl protecting group and tetrazole as the coupling agent. Automated synthesizers that can
quickly and inexpensively synthesize oligos of over 100 nucleotides are commercially available.
14.8.2 Additional antigene agents: RNA interference and ribozymes
RNAi and ribozymes represent two additional approaches to gene silencing/down-regulation with thera-
peutic potential. RNAi is an innate cellular process that achieves silencing of selected genes via an anti-
sense mechanism. It shares many characteristics with the antisense-based approach described above, but
also some important differences, e.g. in the exact mechanism by which the antisense effect is achieved.
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