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J. Biol. Chem., Vol. 283, Issue 4, 2120-2128, January 25, 2008

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Helper-dependent Adenovirus-mediated Short Hairpin RNA Expression in the Liver Activates the Interferon Response^*

Scott R. Witting, Matthew Brown, Romil Saxena, Sarah Nabinger, and Núria Morral^¶1

From the Department of Medical and Molecular Genetics, Department of Pathology, and ^¶Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202

The use of RNA interference has proven to be an effective means to study the function of genes. Constitutive synthesis of small interfering RNA molecules can be accomplished with the use of viral vectors expressing short hairpin RNA (shRNA). Binding of shRNA to the target mRNA promotes transcript degradation. So far, little is known about the effects that shRNA induce in vivo. To determine the feasibility of using helper-dependent adenoviral vectors for expression of shRNA in liver, we have designed an shRNA construct to mouse fabp5 (fatty acid-binding protein 5). Intravenous administration of this vector resulted in 75% silencing of fabp5. Increasing the dose of vector did not result in higher levels of silencing, indicating that there is a threshold for the level of knockdown that can be achieved. Synthesis of high levels of shRNA molecules did not alter the levels of cellular micro-RNA, such as miR-122 and let-7a, suggesting that the exportin-5 pathway was not affected. However, high level shRNA expression resulted in activation of the interferon response. Thus, an important consideration when using shRNA-based vectors in vivo is to closely monitor signs of interferon-stimulated gene expression, since a narrow window exists between gene silencing efficacy and nonspecific effects.

Received for publication, May 21, 2007 , and in revised form, November 16, 2007.

^* This work was supported by National Institutes of Health Grant 1R21 DK069432-01, by the Indiana Genomics Initiative (INGEN) (INGEN of Indiana University is supported in part by Lilly Endowment Inc.), and by the American Heart Association (Postdoctoral Fellowship 0620066Z to S. R. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Medical and Molecular Genetics, Indiana University School of Medicine, 975 West Walnut St., IB 130, Indianapolis, IN 46202. Tel.: 317-278-9039; Fax: 317-274-2387; E-mail: nmorralc{at}iupui.edu.

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