Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice
Adam D. Judge, … , Kevin McClintock, Ian MacLachlan
Adam D. Judge, … , Kevin McClintock, Ian MacLachlan
Published February 23, 2009
Citation Information: J Clin Invest. 2009;119(3):661-673. https://doi.org/10.1172/JCI37515.
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Technical Advance

Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice

Abstract

siRNAs that specifically silence the expression of cancer-related genes offer a therapeutic approach in oncology. However, it remains critical to determine the true mechanism of their therapeutic effects. Here, we describe the preclinical development of chemically modified siRNA targeting the essential cell-cycle proteins polo-like kinase 1 (PLK1) and kinesin spindle protein (KSP) in mice. siRNA formulated in stable nucleic acid lipid particles (SNALP) displayed potent antitumor efficacy in both hepatic and subcutaneous tumor models. This was correlated with target gene silencing following a single intravenous administration that was sufficient to cause extensive mitotic disruption and tumor cell apoptosis. Our siRNA formulations induced no measurable immune response, minimizing the potential for nonspecific effects. Additionally, RNAi-specific mRNA cleavage products were found in tumor cells, and their presence correlated with the duration of target mRNA silencing. Histological biomarkers confirmed that RNAi-mediated gene silencing effectively inhibited the target’s biological activity. This report supports an RNAi-mediated mechanism of action for siRNA antitumor effects, suggesting a new methodology for targeting other key genes in cancer development with siRNA-based therapeutics.

Authors

Adam D. Judge, Marjorie Robbins, Iran Tavakoli, Jasna Levi, Lina Hu, Anna Fronda, Ellen Ambegia, Kevin McClintock, Ian MacLachlan

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Figure 2

In vitro activity of unmodified versus 2′OMe-modified PLK1 and KSP siRNA.

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In vitro activity of unmodified versus 2′OMe-modified PLK1 and KSP siRNA...
Activity of the 2′OMe-modified panels of (A) PLK1424 and (B) PLK773 siRNA. Unmodified PLK1424 or PLK773 siRNA was compared in the Hep3B cell viability assay with the 2′OMe-modified duplexes 1/A, 2/A, 1/B, 2/B, 1/C, and 2/C that comprise the respective 2′OMe sense/AS oligonucleotides (see Table 1). Data show mean viability of triplicate cultures relative to PBS-treated cells and represent 2 independent experiments using SNALP-formulated siRNAs. (C) Cytokine induction by unmodified and 2′OMe PLK1 siRNA in vitro. Murine Flt3L DCs were treated with 5 μg/ml (350 nM) unmodified PLK773 and PLK1424 siRNA duplexes and their constituent sense (S) or AS oligonucleotides or the 2′OMe siRNA duplexes PLK773-1/B (1/B) and PLK1424-2/A (2/A) formulated in SNALP. IFN-α and IL-6 were assayed in culture supernatants at 24 hours. Values represent mean + SD of 3 separate experiments conducted in triplicate cultures. (D and E) Activity of SNALP-formulated KSP2263 siRNA in murine Neuro2a cells. (D) Correlation between KSP mRNA silencing and cell viability relative to PBS control. KSP mRNA was determined by bDNA analysis at 24 hours. Duplicate plates were assessed for cell viability at 72 hours. (E) Activity screen comparing the unmodified KSP2263 siRNA to KSP2263-U/U (U/U), KSP2263-G/U (G/U), and KS2263-G/G (G/G) siRNA duplexes that comprise the respective sense/AS 2′OMe oligonucleotides (see Table 1). SNALP-formulated KSP2263 siRNA were tested in the Neuro2a cell viability assay. Data represent mean ± SD triplicate cultures, relative to PBS treatment.