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Validating The Use of siRNA as a Novel Technique for Cell Specific Target Gene Knockdown in Lung Ischemia-Reperfusion Injury
John Keech, Elizabeth FitzSullivan, Patrick Wolf, Heather Merry, *Michael Mulligan
University of Washington, Seattle, WA
BACKGROUND:Short interfering RNA (siRNA) has been reported as an effective method for knockdown of target genes in vitro and in vivo. However, concerns have been raised regarding design, administration, efficacy, specificity and the immunostimulatory potential of siRNA in vivo. No studies have examined siRNA use in lung ischemia reperfusion injury (LIRI) where siRNA may be well suited for practical pretreatment. Studies have demonstrated effective uptake of intravenously administered siRNA by resident inflammatory cells, including alveolar macrophages (AM). We have also previously demonstrated the central importance of the AM in the development of LIRI. We describe the validation of siRNA as a novel technique for cell specific target gene knockdown in the AM in our model of LIRI.
METHODS:To determine the lowest effective dose of siRNA, Rats received between 1 and 50nM siRNA (TLR-4, TLR-2, MyD88), vector control or saline control intravenously in a lipid vector prior to the IR protocol. Primary cultures of AM, PAEC, and T2P received between 10 and 1000pM siRNA with similar controls prior to our hypoxia reoxygenation (HR) protocol. 3 distinct sequences for TLR-4, TLR-2 and MyD88 siRNA were tested for efficacy. Whole lung homogenates, individual cell populations eluted from lungs, and cell culture lysates were harvested for total protein to assess target protein knockdown by Western blot analysis. Serum from rats and media from cell cultures was assessed for IFNγ and IFNβ production after siRNA administration. Biotin labeled TLR-4 siRNA was used to assess siRNA uptake in vitro and distribution in the lung by IHC.
RESULTS:Rats pretreated with TLR-4 siRNA had a 70-92% reduction in TLR-4 protein expression, and demonstrated significant protection from LIRI. Cell populations eluted from lungs and whole blood from rats treated with TLR-4 siRNA had a >90% reduction in TLR-4 protein expression in AM and no reduction in TLR-4 expression in PAEC, T2P or WBC. Biotin labeled TLR-4 siRNA exclusively localized to AM in the lung. There was >80% knockdown of TLR-4 expression in cultured AM, PAEC, and T2P treated with TLR-4 siRNA. The lowest effective dose of siRNA was determined to be 10nM in vivo and 100pM/well in vitro. There was no significant change in IFN production between TLR-4, TLR-2 or MyD88 siRNA or control rats or AM. All siRNA sequences were able to effectively and specifically knockdown their respective protein expression. (76-95% in vitro).
CONCLUSIONS:We have demonstrated the cell specific uptake of intravenously administered siRNA to the AM in the lung, and employed a series of controls for OTE, validating the efficacy, specificity and immunostimulatory potential with low dose siRNA in our model of LIRI. These results significantly increase the confidence with which the observed phenotype (protection from IR or HR) can be ascribed to knockdown of the target protein, and provide a tool for studying the central role of the AM in the development of LIRI.
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