Constitutive Expression of Short Hairpin RNA in Vivo Triggers Buildup of Mature Hairpin Molecules

Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Human gene therapy (Impact Factor: 3.76). 07/2011; 22(12):1483-97. DOI: 10.1089/hum.2010.234
Source: PubMed


RNA interference (RNAi) has become the cornerstone technology for studying gene function in mammalian cells. In addition, it is a promising therapeutic treatment for multiple human diseases. Virus-mediated constitutive expression of short hairpin RNA (shRNA) has the potential to provide a permanent source of silencing molecules to tissues, and it is being devised as a strategy for the treatment of liver conditions such as hepatitis B and hepatitis C virus infection. Unintended interaction between silencing molecules and cellular components, leading to toxic effects, has been described in vitro. Despite the enormous interest in using the RNAi technology for in vivo applications, little is known about the safety of constitutively expressing shRNA for multiple weeks. Here we report the effects of in vivo shRNA expression, using helper-dependent adenoviral vectors. We show that gene-specific knockdown is maintained for at least 6 weeks after injection of 1 × 10(11) viral particles. Nonetheless, accumulation of mature shRNA molecules was observed up to weeks 3 and 4, and then declined gradually, suggesting the buildup of mature shRNA molecules induced cell death with concomitant loss of viral DNA and shRNA expression. No evidence of well-characterized innate immunity activation (such as interferon production) or saturation of the exportin-5 pathway was observed. Overall, our data suggest constitutive expression of shRNA results in accumulation of mature shRNA molecules, inducing cellular toxicity at late time points, despite the presence of gene silencing.

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Available from: Nuria Morral
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    • "RNA interference (RNAi) is a gene silencing phenomenon which was first discovered in Caenorhabditis elegans triggered by double-stranded RNA (dsRNA) (Lecellier et al., 2005). Several researchers reported hepatotoxicity and fatality induced by ectopic RNAi triggers when they attempted to intravenously inject adeno-associated virus (AAV)-mediated shRNA vectors into mouse models (Ahn et al., 2011; Borel et al., 2011; Martin et al., 2011), which seriously hindered therapeutic RNAi. On the other hand, "
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    • "A longer duration of effect has only been reported in HBV mice receiving adeno-associated virus expressing a short hairpin RNA (shRNA) targeting HBV.30 Although this vector-based approach is promising, lingering concerns over the long-term effects of constitutive shRNA expression remain.31 "
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    • "Furthermore, they also observed shRNA-dependent downregulation of three cellular miRNAs, including liver-specific miR-122, corroborating that shRNA overexpression can adversely perturb the miRNA/RNAi machinery in vivo. In line with this evidence, Ahn et al. [4] noted gradual hepatocyte death in mice treated with shRNA-expressing gutless adenoviral vectors that correlated with the buildup of mature shRNA molecules and the upregulation of miRNA-controlled hepatic genes. "
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