1. ¹Department of Internal Medicine, University of Iowa, Iowa City, Iowa, USA
2. ²Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa, USA
3. ³Department of Neurology, University of Iowa, Iowa City, Iowa, USA

Correspondence: Beverly L. Davidson, Department of Internal Medicine, University of Iowa, 200 Eckstein Medical Research Building, Iowa City, Iowa 52240, USA. E-mail: beverly-davidson@uiowa.edu

Received 2 July 2008; Accepted 23 September 2008; Published online 11 November 2008.

Abstract

RNA interference (RNAi) provides a promising therapeutic approach to human diseases. However, data from recent reports demonstrate that short-hairpin RNAs (shRNAs) may cause cellular toxicity, and this warrants further investigation of the safety of using RNAi vectors. Earlier, in comparing hairpin-based RNAi vectors, we noted that shRNAs are highly expressed and yield an abundance of unprocessed precursors, whereas artificial microRNAs (miRNAs) are expressed at lower levels and are processed efficiently. We hypothesized that unprocessed shRNAs arise from the saturation of endogenous RNAi machinery, which poses likely a burden to cells. In this study, we tested that hypothesis by assessing the relative effects of shRNAs and artificial miRNAs on the processing and function of miRNAs. In competition assays, shRNAs disrupted miRNA biogenesis and function, whereas artificial miRNAs avoided this interference even when dosed to silence as effectively as shRNAs. We next compared the safety of these vectors in mouse cerebella, and found that shRNAs cause Purkinje cell neurotoxicity. By contrast, artificial miRNA expression was well tolerated, resulting in effective target gene silencing in Purkinje cells. These findings, together with data from earlier work in mouse striata, suggest that miRNA-based platforms are better suited for therapeutic silencing in the mammalian brain.