Optisense® screening rapidly identifies optimal target sites for
therapeutic oligonucleotides
Zhidong Chen, Christopher Clemens, Richard Koehn, Robert Risley,
Duane Ruffner, Lauren Smiley. Genta Incorporated, Salt Lake City, UT.
Background: BCL-2 is a member of the protein family whose function is
to control apoptosis. BCL-2 represses apoptosis and is expressed in a
large number of cancer types. The well-demonstrated ability of this
protein to impart resistance against most cancer therapies makes BCL-
2 one of the most important molecular targets in oncology. In a
recently completed, randomized Phase 3 trial, Genasense™ (oblimersen
sodium) plus chemotherapy significantly increased antitumor responses
as well as progression-free survival in patients with advanced
malignant melanoma, a notoriously chemo-resistant disease. We have
used a novel screening method, OptiSense®, to evaluate target
identification and optimization of all forms of inhibitory
oligonucleotides, using BCL-2 as a target gene and oligonucleotide
G3139, which is the research antisense molecule of Genasense™, as a
positive control.
Methods: OptiSense® is a deletion generation, cloning, and expression
method that produces a library of independent clones containing
ordered oligonucleotide fragments 14 or 18 bases in length from the
cDNA of any target gene. An expressed anti-BCL-2 oligonucleotide
library was generated and functionally screened in mammalian cells to
identify the optimal anti-BCL-2 target sites. We then synthesized
oligonucleotides targeted to these sites, using a variety of chemical
compositions and oligonucleotide variants, including all-
phosphorothioate antisense DNA, siRNA, and single-stranded RNA. They
were then evaluated both in vitro and in vivo for anti-BCL-2 and anti-
tumor activity versus G3139. In addition, the anti-BCL-2
oligonucleotides were delivered both as free oligonucleotides and as
oligonucleotides formulated in PolyBus® (a proprietary
polyethyleneglycol /polyethyleneimine conjugate delivery vehicle for
nucleic acids).
Results: We identified several anti-BCL-2 oligonucleotides with
equivalent or improved inhibitory activity against BCL-2 versus
G3139, measured at both the mRNA and protein levels in multiple
tissue culture lines. These new anti-BCL-2 oligonucleotides also
showed equivalent or increased anti-tumor activity versus G3139 in
multiple nude mouse/human tumor xenograft models. The most promising
of these oligonucleotides was GS36518, which inhibited the growth of
chemo-resistant human ovarian tumor xenograft and other tumor models
in nude mice.
Conclusions: The OptiSense® method can be used to identify optimal
inhibitory sequences against any gene of interest within living
cells. In contrast to in silico predictions and cell-free mappings,
OptiSense® identified target sites are biologically relevant. These
target sites can be used for gene inhibition via antisense, ribozyme,
siRNA, and microRNA mechanisms.
Presenter: Zhidong Chen
Affiliation: Genta Incorporated, Salt Lake City, UT; E-mail:
brown@genta.com
Copyright © 2004 American Association for Cancer Research. All rights
reserved. Citation information: Proceedings of the AACR, Volume 45,
March 2004. |
