Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver

Abstract

shRNA overexpression from viral gene therapy vectors can trigger cytotoxicity leading to organ failure and lethality in mice and rats. This process likely involves saturation of endogenous cellular RNAi factors including exportin-5 (Xpo-5). Here, we have shown that Xpo-5 overexpression enhanced shRNA efficiency in the liver of adult mice but increased hepatotoxicity. We identified the 4 members of the human Argonaute (Ago) protein family as downstream factors involved in saturation of endogenous cellular RNAi, all of which were able to interact with shRNAs in cells and mice. In Ago/shRNA coexpression studies, Ago-2 (Slicer) was the primary rate-limiting determinant of both in vitro and in vivo RNAi efficacy, toxicity, and persistence. In adult mice, vector-based Ago-2/Xpo-5 coexpression enhanced U6-driven shRNA silencing of exogenous and endogenous hepatic targets, reduced hepatotoxicity, and extended RNAi stability by more than 3 months. Use of weaker RNA polymerase III promoters to minimize shRNA expression likewise alleviated in vivo toxicity and permitted greater than 95% persistent knockdown of hepatitis B virus and other transgenes in mouse liver for more than 1 year. Our studies substantiate that abundant small RNAs can overload the endogenous RNAi pathway and reveal possible strategies for reducing hepatotoxicity of short-and long-term clinical gene silencing in humans.

Caspase-3 and GFAP as early markers for apoptosis and astrogliosis in shRNA-induced hippocampal cytotoxicity

“…hcn and luc), but were not apparent in animals treated with rAAVs encoding only eGFP. Cytotoxic effects of shRNAs have previously been shown to be caused by overloading of the endogenous miRNA machinery (Börner et al, 2013;Grimm et al, 2006Grimm et al, , 2010McBride et al, 2008;Yi et al, 2005). These observations were corroborated by the pronounced shRNA dose dependency of cytotoxicity (Grimm, 2011).…”

“…Certain aspects of shRNA-induced cytotoxicity have been described in previous studies (Börner et al, 2013;Bridge et al, 2003;Grimm et al, 2010;Sledz et al, 2003;Yi et al, 2005). Notably, shRNA-induced neurotoxicity is affected by various factors, such as shRNA sequence, shRNA concentration applied, means of application (e.g.…”

A universal TaqMan-based RT-PCR protocol for cost-efficient detection of small noncoding RNA

“…That might be explained by strand displacement activities of the MMuLV reverse transcriptase used in our assays that were reported previously, at least for DNA-DNA and RNA-DNA hybrids (Kelleher and Champoux 1998); although the method can distinguish between pre-miRNA and mature miRNA, the stem-loop primers likely are not able to discriminate singlestranded and double-stranded stages of processed mature miRNA, i.e., the stages from Dicer cleavage, passenger strand cleavage, to RISC loading complex (RLC)-and RNA-induced silencing complex (RISC)-associated RNA. Since the availability of Argonauts as the active component of RISC is known as the limiting factor in RNAi (Grimm et al 2010;Winter and Diederichs 2011), the method must be applied with care in cases of overloading the RNAi pathway with miRNA, small hairpin (sh)RNA, or siRNA. Under these conditions, the results will not necessarily correlate with the RNAi effector quantity.…”

RNAi in Xenopus: look before you leap

“…It would be interesting to see whether this conjecture can be confirmed by microarray analysis, to see whether miR-427 targets are nonspecifically up-regulated by diverse siRNAs, similar to the accumulation of miR-430 target transcripts in Dicer-mutant fish embryos (Giraldez et al 2006). In any case, the saturability of endogenous Ago pools mirrors observations in other systems where elevation of Ago proteins can increase the accumulation of miRNAs or endosiRNAs (Yigit et al 2006;Diederichs and Haber 2007) and enhance on-target suppression (Diederichs et al 2008;Grimm et al 2010).…”