title: Constitutive expression of short hairpin RNA in vivo triggers buildup of mature hairpin molecules.
abstract: 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.
authors.affiliation: Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: M
authors.initials: M
authors.lastname: Ahn
authors.forename: S R
authors.initials: SR
authors.lastname: Witting
authors.forename: R
authors.initials: R
authors.lastname: Ruiz
authors.forename: R
authors.initials: R
authors.lastname: Saxena
authors.forename: Núria
authors.initials: N
authors.lastname: Morral
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D050556:Fatty Acid-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D020869:Gene Expression Profiling; D020868:Gene Silencing; D005822:Genetic Vectors / Q000008:administration & dosage*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D008099:Liver / Q000378:metabolism* / Q000473:pathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D051780:Sterol Regulatory Element Binding Protein 1 / Q000037:antagonists & inhibitors* / Q000235:genetics; D014161:Transduction, Genetic
chemical_list: D015415:Biomarkers; C082667:Fabp5 protein, mouse; D050556:Fatty Acid-Binding Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C497388:Srebf1 protein, mouse; D051780:Sterol Regulatory Element Binding Protein 1