Abstract
Background: We have recently introduced an intelligent RNA expression device (iRed), comprising the minimum essential components needed to transcribe short hairpin RNA (shRNA) in cells. Use of iRed efficiently produced shRNA molecules after transfection into cells and alleviated the innate immune stimulation following intravenous injection. Methods: To study the usefulness of iRed for local injection, the engineered iRed encoding luciferase shRNA (Luc iRed), complexed with cationic liposomes (Luc iRed/liposome-complexes), was intrapleurally injected into an orthotopic mesothelioma mouse model. Results: Luc iRed/liposome-complexes markedly suppressed the expression of a luciferase marker gene in pleurally disseminated mesothelioma cells. The suppressive efficiency was correlated with the expression level of shRNA within the mesothelioma cells. In addition, intrapleural injection of iRed/liposome-complexes did not induce IL-6 production in the pleural space and consequently in the blood compartment, although plasmid DNA (pDNA) or dsDNA (the natural construct for iRed) in the formulation did. Conclusion: Local delivery of iRed could augment the in vivo gene silencing effect without eliciting pronounced innate immune stimulation. Our results might hold promise for widespread utilization of iRed as an RNAi-based therapeutic for intracelial malignant cancers.
Highlights
RNA interference (RNAi), a biological process in which RNA molecules inhibit gene expression or translation, has emerged as a powerful tool for studying gene function and holds promise for the development of new gene silencing therapies [1,2]
As a novel approach to alleviate the innate immune stimulation of plasmid DNA (pDNA) molecules, we have developed a new artificial DNA construct, i.e., “intelligent RNA expression device”, and studied its ability to induce RNAi gene-silencing [10]. iRed contains the minimum number of components needed for short hairpin RNA (shRNA) production in cells, including a U6 promoter and an shRNA-encoding region, in which cognate 4’-thio derivatives of thymine (T), guanine (G), cytosine (C) or adenine (A) nucleotide units were substituted for natural nucleotides [11,12]
To expand the usefulness of iRed, we studied the effectiveness of engineered dSC iRed, in which cytosine (C) nucleotide unit was substituted by cognate 40 -thio derivatives, encoding luciferase shRNA (Luc iRed) given by local intracelial injection in a luciferase-expressing orthotopic murine model of pleural mesothelioma
Summary
RNA interference (RNAi), a biological process in which RNA molecules inhibit gene expression or translation, has emerged as a powerful tool for studying gene function and holds promise for the development of new gene silencing therapies [1,2]. RNA (siRNA) and short hairpin RNA (shRNA), produced from plasmid DNA (pDNA), have gained considerable interest as tools for gene silencing [3]. ShRNA, produced from pDNA, has lower expression efficiency. Use of iRed efficiently produced shRNA molecules after transfection into cells and alleviated the innate immune stimulation following intravenous injection. Results: Luc iRed/liposome-complexes markedly suppressed the expression of a luciferase marker gene in pleurally disseminated mesothelioma cells. Intrapleural injection of iRed/liposome-complexes did not induce IL-6 production in the pleural space and in the blood compartment, plasmid DNA (pDNA) or dsDNA (the natural construct for iRed) in the formulation did. Conclusion: Local delivery of iRed could augment the in vivo gene silencing effect without eliciting pronounced innate immune stimulation. Our results might hold promise for widespread utilization of iRed as an RNAi-based therapeutic for intracelial malignant cancers
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