Antisense Oligonucleotides as Potential Drugs
annealing of a randomized oligodeoxynu-
cleotide preferably to the native RNA
and subsequent cleavage by endogenous
RNase H is the nearest to the intracellu-
lar situation and yet is relatively simple
to perform. In any event, in most cases
a number of AS-ODNs will be synthe-
sized to select the most active. Depending
on the laboratory, this number can be up
to twenty.
One of the problems for the antisense strat-
egy is the intracellular availability of the
negatively charged oligonucleotides with
a molecular weight of several thousand
daltons. ODNs are usually not taken up
efFciently in cell culture. Electroporation
is one way of delivering the ODNs to the
cell, but this method is not of general
applicability. The most commonly used
strategy for cell culture experiments is the
complexation of the oligonucleotides with
synthetic lipsosomes with a positive charge
on the surface. A large variety of these com-
pounds are commercially available. These
carriers by themselves are usually toxic to
the cell because of the positive charges, but
once covered by the ODN this cytotoxicity
is in most cases abolished. Transfection
efFciencies can widely differ, but this can
be monitored by cotransfection with a flu-
orescent ODN. In most cases, the ODNs
are trapped to a considerable degree in the
endosome so that only a fraction is located
in the cytoplasm or in the nucleus, places
where they should be operative. Most in-
teresting is the observation that uptake of
in vivo
, that is, in animal models or
even man, does not require such carriers.
The delivery there is simply achieved, for
example, by subcutaneous or intravenous
infusion. This difference for
in vitro
in vivo
cellular uptake signiFes a consider-
able difference between the two systems,
which is not understood at present. What-
ever the cause of it, it should caution for
extrapolating from the
in vitro
results to
relevance. Independent of the method
of delivery, considerable efforts are spent
on directing the ODNs speciFcally to the
desired cell or organ type.
Uptake of the ODNs with a neutral back-
bone such as the morpholinos or the PNAs
cannot rely on cationic carriers because of
lack of binding. They require cell scraping,
injection, or, as shown for PNA, conju-
gation with cationic peptides to facilitate
cellular uptake. At present, this presents
a certain barrier for wide application, in
particular, for medical purposes.
The literature on the application of ODNs
for the inhibition of gene expression
is vast.
We have summarized in Table 1 those
examples in which the technique has ad-
vanced to clinical-phase trials, although
this collection is probably not complete.
Not surprisingly, considering the enor-
mous expense involved, these investiga-
tions are almost exclusively conducted by
companies. The only notable exception is
the study by the laboratory of A. Gewirtz
on CML for
ex vivo
Not all the examples listed can be dis-
cussed here, so some comments must
sufFce. Common to all ODNs used in
the clinical trials is one aspect of their
chemical nature in that they all are phos-
phorothioate-containing ODNs. The Frst
approved antisense drug was developed
by ISIS, ±omivirsen or Vitravene, a trade
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