Artificial MicroRNAs as siRNA Shuttles: Improved Safety as Compared to shRNAs In vitro and In vivo

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.

Incentive Learning Underlying Cocaine-Seeking Requires mGluR5 Receptors Located on Dopamine D1 Receptor-Expressing Neurons

“…This is not the case for the mGluR5 KD-D1 mice, where maturation of short RNAs is normal. Most likely, previously reported problems were caused by the use of tools resulting in very high levels of short RNAs, such as strong RNA polymerase III promoters or the use of shRNAs instead of artificial microRNAs (Boudreau et al, 2009). Another potential problem is off-target effects.…”

MECHANISMS IN ENDOCRINOLOGY: MicroRNA in diagnostics and therapy of thyroid cancer

“…The biological impact of upregulated microRNAs can be abolished with specific inhibitors or anti-miRs, which are synthetic molecules that prevent binding of a miR to its targets. Lowered levels of a microRNA can be reversed by its directed upregulation, accomplished through the delivery of synthetic mature microRNA mimics, as in the synthetic RNA (siRNA) technology (57,58), or the delivery of primary microRNAs (shmiR) or microRNA precursors (shRNA) expressed from plasmids (59,60,61).…”

Recent Advances in RNA Interference Therapeutics for CNS Diseases

“…shRNAs are typically expressed from strong Pol III promoters (such as U6 or H1), while artificial miRNAs can be expressed from pol II or pol III promoters. While shRNAs may have more potent silencing capability, they are often expressed at very high levels and can saturate the RNAi machinery, which disrupts endogenous miRNA processing and can induce toxicity [18,19]. Artificial miRNAs, however, are generally safer and less toxic, and they do not appear to disrupt endogenous miRNA processing [18][19][20][21][22][23].…”