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Effective RNAi-mediated gene silencing without interruption of the endogenous microRNA pathway

Abstract

Systemic administration of synthetic small interfering RNAs (siRNAs) effectively silences hepatocyte gene expression in rodents and primates1,2,3. Whether or not in vivo gene silencing by synthetic siRNA can disrupt the endogenous microRNA (miRNA) pathway remains to be addressed. Here we show that effective target-gene silencing in the mouse and hamster liver can be achieved by systemic administration of synthetic siRNA without any demonstrable effect on miRNA levels or activity. Indeed, siRNA targeting two hepatocyte-specific genes (apolipoprotein B and factor VII) that achieved efficient (80%) silencing of messenger RNA transcripts and a third irrelevant siRNA control were administered to mice without significant changes in the levels of three hepatocyte-expressed miRNAs (miR-122, miR-16 and let-7a) or an effect on miRNA activity. Moreover, multiple administrations of an siRNA targeting the hepatocyte-expressed gene Scap in hamsters achieved long-term mRNA silencing without significant changes in miR-122 levels. This study advances the use of siRNAs as safe and effective tools to silence gene transcripts in animal studies, and supports the continued advancement of RNA interference therapeutics using synthetic siRNA.

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Figure 1: Northern blot analysis of mature miRNAs reveals no detectable differences among siRNA treatment groups.
Figure 2: Liver mRNA levels of miR-122 targets are unaffected by siRNA treatment.
Figure 3: Long-term silencing of Scap in hamster liver by repeat administration of siRNA does not affect miR-122 levels.

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Acknowledgements

We thank A. Wetzel, M. Jayaraman, K. N. Jayaprakash, K. G. Rajeev, L. Nechev and G. Wang for assistance in chemistry; R. Dorkin for liposome preparation; M. Duckman for assistance with the figures; and J. Maraganore for critical advice on the manuscript. M. Spranger is supported by CC-SPMD/ETH. M.G., R.L. and D.G.A. are supported by an NIH grant.

Author Contributions Experimental work was performed by M.J., R.C., A.A., M.G., Y.-A.M. and M. Spranger. Experimental design and critical advice was provided by M.J., R.C., A.A., P.H., J.S., H.-P.V., M.M., M. Stoffel, R.L., D.G.A., J.D.H., V.K. and D.B. The paper was written by D.B.

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Correspondence to David Bumcrot.

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Competing Interests Statement: The following authors are employees of Alnylam Pharmaceuticals, Inc., or Alnylam Europe AG, which develops therapeutics based on siRNAs: M. John, R. Constien, A. Akinc, P. Hadwiger, J. Soutschek, H.-P. Vornlocher, M. Manoharan, V. Koteliansky and D. Bumcrot. In addition, M. Stoffel, R. Langer and D. G. Anderson are consultants to Alnylam. The remaining authors declare no competing interests.

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John, M., Constien, R., Akinc, A. et al. Effective RNAi-mediated gene silencing without interruption of the endogenous microRNA pathway. Nature 449, 745–747 (2007). https://doi.org/10.1038/nature06179

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