Antisense Oligonucleotides as Potential Drugs
413
This process has now been extended to
many organisms, mainly in cell culture
but also in a few animal model stud-
ies for gene function analysis. It requires
double-stranded oligoribonucleotides 21
nucleotides in length (called
siRNAs
)o
f
which one strand is completely com-
plementary to the mRNA target. Upon
binding of this strand to the mRNA in
a RNA induced silencing complex (RISC)
complex, the target is cleaved opposite
the oligoribonucleotide. The breakthrough
in the Feld came by the observation
that the oligonucleotides can be added
exogenously, thus facilitating wide appli-
cation. They can be chemically synthesized
or obtained by
in vitro
transcription to
be transfected with the aid of a carrier
as detailed earlier. Alternatively, vectors
can be constructed for stable transfec-
tion where the sequence of the oligonu-
cleotides is positioned, most often behind
the U6 promoter, for intracellular tran-
scription. The inhibition by the siRNAs
is not very dependent on the secondary
structure of the mRNA. Usually three
constructs are sufFcient to identify the
most active oligonucleotide pair, thus
much fewer than that required for the
AS-ODNs as described earlier. Also, in
general, the inhibition achieved is very
satisfactory.
8
Ribozymes
Although not normally considered in the
context of the antisense methodology, ri-
bozymes fall into this category as the basis
for their interaction with RNA is based on
the sense–antisense principle. The fun-
damental difference between the classical
antisense method and that of RNA interfer-
ence is that ribozymes have the inherent
catalytic power to cleave the target RNA
rather than to have to rely on cellular pro-
teins for this step. Ribozymes will not be
discussed here in depth, but two recent
reviews give an excellent account of the
present understanding of the various types
of ribozymes. Some clinical trials are ongo-
ing that are predominantly directed against
Hepatitis C (Heptazyme), the VEG± recep-
tor (Angiozyme), and the human epider-
mal growth factor HER2 (Herzyme).
9
Conclusion
This review demonstrates that the anti-
sense methodology is not as simple as orig-
inally anticipated. As research progresses,
more and more insight is gained into the
complexity of responses induced by the
insertion of an oligonucleotide in a cell.
Given these problems, it is a great achieve-
ment that quite a few ODNs are now in
clinical trials. It provides encouragement
to pursue this line of development further.
With the knowledge available from past
experience, it is expected that the pace of
the development of oligonucleotide-based
drugs will accelerate.
See also
Bioorganic Chemistry.
Bibliography
Books and Reviews
Agrawal, S., Kandimalla, E. (2001) Antisense
and/or immunostimulatory oligonucleotide
therapeutics,
Curr. Cancer Drug Targets
1
,
197–209.
Bennet, C.±.
(1998)
Use
of
Cationic
Lipid
Complexes
for
Antisense
Oligonucleotide
Delivery, in: Stein, C.A., Krieg, A.M. (Eds.)
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