Gene expression: RNA interference in adult mice

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Nature 418, 38 - 39 (2002); doi:10.1038/418038a  


Gene expression: RNA interference in adult mice


RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.


Small interfering RNAs (siRNAs) mimic intermediates in the RNA-interference (RNAi) pathway and can silence genes in somatic cells without activating non- specific suppression by double-stranded RNA-dependent protein kinase1. To investigate whether siRNAs also inhibit gene expression in vivo, we used a modification of hydrodynamic transfection methods2-4 to deliver naked siRNAs to the livers of adult mice. Either an siRNA derived from firefly luciferase or an unrelated siRNA was co-injected with a luciferase-expression plasmid (for construct description and sequences, see supplementary information). We monitored luciferase expression in living animals using quantitative whole-body imaging5 (Fig. 1a, c), and found that it was dependent on reporter-plasmid dose (results not shown).


Figure 1 RNA interference in adult mice.   Full legend

High resolution image and legend (48k)
In each experiment, serum measurements of a co-injected human -1 antitrypsin (hAAT) plasmid6 served to normalize transfection efficiency and to monitor non-specific translational inhibition. Average serum concentrations of hAAT after 74 h were similar in all groups.

Our results indicate that there was specific, siRNA-mediated inhibition of luciferase expression in adult mice (P < 0.0115) and that unrelated siRNAs had no effect (P < 0.864; Fig. 1a, b). In 11 independent experiments, luciferase siRNAs reduced luciferase expression (as judged by emitted light) by an average of 81% ( 2.2%). These findings indicate that RNAi can downregulate gene expression in adult mice.

As RNAi degrades respiratory syncitial virus RNAs in culture7, we investigated whether RNAi could be directed against a human pathogenic RNA expressed in a mouse, namely that of hepatitis C virus (HCV). (Infection by HCV, an RNA virus that infects 1 in 40 people worldwide, is the most common reason for liver transplantation in the United States and Europe.) We fused the NS5B region (non-structural protein 5B, viral-polymerase-encoding region) of this virus with luciferase RNA and monitored RNAi by co-transfection in vivo. An siRNA targeting the NS5B region reduced luciferase expression from the chimaeric HCV NS5B protein–luciferase fusion by 75% ( 6.8%; 6 animals per group). This result suggests that it may be feasible to use RNAi as a therapy against other important human pathogens.

Although our results show that siRNAs are functional in mice, delivery remains a major obstacle. Unlike siRNAs, functional small-hairpin RNAs (shRNAs) can be expressed in vivo from DNA templates using RNA polymerase III promoters8, 9; they are as effective as siRNAs in inducing gene suppression. Expression of a cognate shRNA (pShh1-Ff1; see supplementary information) inhibited luciferase expression by up to 98% ( 0.6%), with an average suppression of 92.8% ( 3.39%) in three independent experiments (Fig. 1c, d). An empty shRNA-expression vector had no effect (results not shown); reversing the orientation of the shRNA (pShh1-Ff1rev) insert prevents gene silencing because it alters the termination by RNA polymerase III and generates an improperly structured shRNA. These findings indicate that plasmid-encoded shRNAs can induce a potent and specific RNAi response in adult mice.

RNAi may find application in functional genomics or in identifying targets for designer drugs. It is a more promising system than gene-knockout mice because groups of genes can be simultaneously rendered ineffective without the need for time-consuming crosses. Gene therapy currently depends on the ectopic expression of exogenous proteins; however, RNAi may eventually complement this gain-of-function approach by silencing disease-related genes with DNA constructs that direct the expression of shRNAs. Our method of RNAi delivery could also be tailored to take advantage of developing viral and non-viral gene-transfer vectors in a clinical context.

Supplementary information accompanies this paper.


ANTON P. MCCAFFREY*, LEONARD MEUSE*, THU-THAO T. PHAM*, DOUGLAS S. CONKLIN†, GREGORY J. HANNON† & MARK A. KAY*
* Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, California 94305-5208, USA
† Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York

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