406
Antisense Oligonucleotides as Potential Drugs
expression, which will be discussed later
in a separate section.
The antisense oligodeoxynucleotide ap-
proach has been reviewed recently. A very
thoughtful review of the different types of
inhibition of gene expression with oligonu-
cleotides has also appeared recently.
2
Chemical Modifcation oF Oligonucleotides
2.1
Unmodifed Oligonucleotides
Natural oligodeoxynucleotides are widely
used as tools in molecular biology; how-
ever, they have serious limitations in
their application as therapeutics for the
sequence-speciFc inhibition of gene ex-
pression.Sucholigonucleotideswithanat-
ural phosphodiester internucleoside link-
age are degraded in serum within minutes
to a few hours, mainly by the action
of fast-cleaving 3
0
-exonucleases that are
accompanied by slower-cleaving endonu-
cleases. In certain tissues, a signiFcant
5
0
-exonuclease activity has also been ob-
served. However, oligonucleotides can be
protected against nucleolytic cleavage by
packaging into liposomes or nanoparticles
that, at the same time, serve as carriers for
in vivo
application of these compounds as
discussed below.
2.2
Phosphorothioates
Because of the nuclease sensitivity of
ODNs, the major aim from the begin-
ning was to strengthen the stability against
such degradation by chemical modiFca-
tion. The Frst such modiFcation was the
introduction of phosphorothioate groups
as internucleotidic linkages, which has
been quite successful so that most anti-
sense oligonucleotides (AS-ODNs) in use
are the phosphorothioate analogues. This
modiFcation indeed enhances the stability
of the ODNs against enzymatic degrada-
tion and at the same time is compatible
with activation of RNase H. Thus, these
analogues look ideal but unfortunately
they have some drawbacks. Thus, the
thermal stability of the complex formed
with the target RNA is not as stable as
that formed with an unmodiFed oligonu-
cleotide. Moreover, for reasons that are
not well understood, the phosphorothioate
ODNs can have a much higher afFnity to
certain proteins such as those responsi-
ble for polyanion binding. This can result
in undesired side effects often associated
with cytotoxicity. Thus, reducing the num-
ber of phosphorothioates in an AS-ODN is
advisable and will be discussed below.
As replacement of one of the non-
bridging oxygens of the internucleotidic
phosphate results in a chiral phospho-
rus, the chemical synthesis of the ODN
results in a mixture of diastereomers un-
less efforts are undertaken to render the
synthesis stereospeciFc, which is possible
but not routinely done. There does not
seem any particular advantage for either
isomer in terms of the combination of
thermal stability and RNase H activation
over the mixture.
In summary, the advantages of the
phosphorothioate modiFcation outweighs
the disadvantages at least up to now,
making them a Frst generation of AS-
ODNs.
2.3
Alkylphosphonates
Rather early, it was assumed that the
negative charge of the phosphate back-
bone of the ODNs might interfere with
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