Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

Abstract

As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.

Identification of RISC-Associated Adenoviral MicroRNAs, a Subset of Their Direct Targets, and Global Changes in the Targetome upon Lytic Adenovirus 5 Infection

“…Unlike in the situation where AGO proteins are overexpressed, in the situation where AGO proteins are limiting and the RISCs are already occupied with cellular miRNAs, de novo-synthesized mivaRNAs are likely to become less readily incorporated into RISCs. In analogy, overexpression of AGO2 has been demonstrated to abrogate saturation of RISCs, consequently facilitating RISC incorporation of exogenous siRNAs or shRNAs by abolishing the competition with cellular miRNAs (41)(42)(43)(44). Thus, our data indicate that RISC occupation by mivaRNAs appears to have been overestimated in the past, because under physiologically more relevant conditions, cellular miRNAs are still dominating over mivaRNAs in endogenous RISCs.…”

In Vivo Hepatic Reprogramming of Myofibroblasts with AAV Vectors as a Therapeutic Strategy for Liver Fibrosis

“…We focused on AAV capsids effective in transducing fibroblasts or other mesenchymal cell types, including the naturally occurring AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9 capsids (Blankinship et al, 2004; Di Pasquale et al, 2003; Gao et al, 2004; Grimm and Kay, 2003; Rutledge et al, 1998) and the engineered capsids AAV1P4 (seven amino acid re-targeting peptide displayed in an exposed capsid loop) (Borner et al, 2013), AAV2(Y444,500,730F) (mutation of three exposed tyrosines to phenylalanines) (Li et al, 2010) and AAV-DJ (chimera of AAV2/8/9) (Grimm et al, 2008). After treating wildtype mice with 12 doses of CCl4 we intravenously injected them with 1 × 10 11 viral genomes of an AAV-EYFP vector pseudotyped with one of the nine capsids and analyzed the mouse organs after seven additional doses of CCl4.…”

Alleviation of off-target effects from vector-encoded shRNAs via codelivered RNA decoys

“…All cells were cultured under standard growth conditions [5% (vol/vol) CO 2 , 37°C] and passaged upon confluency. For transfections, HEK293T cells were grown to 70-90% confluency and transfected using polyethylenimine (PEI) as described elsewhere (29). Huh7 cells were grown to 90-95% confluency and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's guidelines.…”

“…Three days posttransfection, cells were harvested using a cell scraper and were lysed by repeated freezethaw cycles. scAAV vector particles were purified via iodixanol (Optiprep; Sigma) density gradient ultracentrifugation for 2 h at 50,000 rpm at 4°C in a Beckman 70.1Ti rotor and a Beckman L-90K ultracentrifuge as described previously (29). scAAV vector genome titers were determined by quantitative PCR against a plasmid standard curve (29,70).…”

“…Nonetheless, several groups have elucidated shRNA design rules that promise a bias toward one shRNA strand, including asymmetric hairpin configurations with mismatches or bulges at the 5′ end of the antisense strand or bioinformatical preselection of shRNAs with minimal off-targeting potential within a given cellular genome (25)(26)(27)(28). Furthermore, various laboratories, including ours, have devised shRNA variants that skip Dicer processing and are loaded directly into Argonaute-2, which then cleaves one of the two strands and predominantly leaves the other as a guide (29)(30)(31)(32)(33)(34).…”