Abstract
A library of 2'-methoxyethyl-modified antisense oligonucleotides (2'MOE ASO) targeting 1,510 different genes has been developed, validated, and used to identify cell cycle regulatory genes. The most effective molecular target identified was Eg5 (kinesin-like-1), which when inhibited gave the largest increase in 4N DNA in various tumor cells. The Eg5 ASO reduced Eg5 levels, inhibited proliferation, increased apoptosis, and altered the expression of other cell cycle proteins, including survivin and Aurora-A. To examine the therapeutic utility of the Eg5 ASO, the compound was also evaluated in xenograft models. Treatment with Eg5 ASO produced a statistically significant reduction of tumor growth, reduction in Eg5 expression in the tumors, and changes in histone phosphorylation, consistent with a loss of Eg5 protein expression. These data show, for the first time, the utility of a 2'MOE ASO library for high-throughput cell culture-based functional assays and suggest that an Eg5 ASO also has potential in a therapeutic strategy.
Highlights
Strategies developed to inhibit cell cycle progression, and proliferation, have led to the successful implementation of numerous therapeutics for various cancers
A minimum of 70% target mRNA reduction for each lead ASO was selected as an acceptance criterion to move an ASO into the library, and ASOs that were within the amplicon of the primer/probe set were not used for the library
We have developed and used a library of validated ASOs to identify genes involved in regulating G2-M cell cycle progression
Summary
Strategies developed to inhibit cell cycle progression, and proliferation, have led to the successful implementation of numerous therapeutics for various cancers. The microtubule-targeting tubulin-polymerizing agents, paclitaxel and taxotere, are widely used to treat human epithelial cancers [1] This class of compounds typically arrests cancer cells in G2-M phase followed by induction of apoptosis. A recent example of a molecular-targeted therapy directed against a cell cycle regulating protein is the development of a small-molecule inhibitor of Aurora kinases which displayed antitumor activity against a variety of tumor models [4]. This and other targeted approaches are encouraging, identifying the most effective gene targets that will result in mitotic arrest is still a
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