In:
Molecular Cancer Therapeutics, American Association for Cancer Research (AACR), Vol. 8, No. 12_Supplement ( 2009-12-10), p. C148-C148
Abstract:
Background: Since oligonucleotides do not penetrate cells well, most RNA-based therapies, such as antisense molecules and siRNAs use transfection conditions in tissue culture and delivery technologies in animals to facilitate entry of oligonucleotides into target cells. However, such vehicles are known to substantially alter cellular function and thereby significantly confound analyses. We have been developing LNA-based RNA antisense molecules to key cancer targets including survivin, HIF-1α, androgen receptor, GLI2, β-catenin, HER3, p110 alpha, and HSP27. LNA-based oligonucleotides were choosen since these molecules are highly stabile in plasma, have extraordinary high affinity to mRNA, and potently silence mRNA in cells under transfection conditions (IC50 in the high picomolar - low nanomolar range). We now report that LNA based RNA antagonist, when used in the high nanomolar - low micromolar range readily down modulate mRNA and protein of interests in multiple cell lines without any delivery means. Material and Methods: In vitro, the ability of the antagonists to down modulate mRNA and cell growth was evaluated by qRT-PCR and MTS respectively. Cellular localization of the antagonists was evaluated by examining a FAM-labeled HER3 antagonist EZN-3920 under fluorescent microscope. In vivo, HER3 mRNA down-modulation and protein levels in tumors, which were grown on the flank of nude mice, were evaluated after intravenous administration of the antagonists. Results: The down-modulation of mRNA in tissue culture-treated cells was quick, robust, long lasting, and specific followed by potent protein inhibition. Importantly, we show that mechanisms underlying the down-modulation correlated with nuclear localization of the LNA-ASO, suggesting that RNas H was the primary mechanism involved in target inhibition. Using this simple and effective approach, we were able to demonstrate that HER3, GLI2, and β-catenin were likely crucial growth and survival factors for certain cancer cells. Conclusions: LNA-based RNA antagonists down-modulate target mRNA and protein in the absence of any agent to facilitate cell penetration. Such antagonists represent an attractive option for rapid, simple, and specific determination of gene function, target validation, and identification of drug-sensitive tumors for further in vivo evaluation. Beyond this, these results helps validate the use of intravenously administered LNA oligonucleotides simply prepared in saline, as is being done in on-going clinical trials with LNA-based RNA antagonists to HIF-1α and survivin. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C148.
Type of Medium:
Online Resource
ISSN:
1535-7163
,
1538-8514
DOI:
10.1158/1535-7163.TARG-09-C148
Language:
English
Publisher:
American Association for Cancer Research (AACR)
Publication Date:
2009
detail.hit.zdb_id:
2062135-8
SSG:
12
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