Adeno-associated virus-mediated microRNA delivery and therapeutics

Abstract

MicroRNAs (miRNAs) are 20 to 24 nt long, single-stranded RNAs that repress gene expression. Dysregulation of miRNA expression is associated with many human diseases. Modulating the level of endogenous miRNA alters gene profiling and can achieve therapeutic benefits. Here the authors review currently used methods of altering miRNA activity in vivo. They focus on the delivery of miRNAs and miRNA inhibitors using recombinant adeno-associated virus (rAAV). In general, rAAV-mediated miRNA inhibition or overexpression provides a simple, efficient, and informative way to study miRNA function in mammals. This method also provides the opportunity to explore potential miRNA therapeutics for many diseases.

Figure 1

(A) Sponge is tandem repeats of miRNA binding site after reporter gene driven by Pol II promoters. The imperfect paring between microRNA and sponge is diagrammed for miR-122. (B) Tough decoy RNAs (TuDs) contain two single-stranded miRNA binding sites, flanked by double-stranded stems that enhance stability and promote nuclear export. U6 promoter is used for high level expression of the miRNA inhibitors.

Figure 2

Validation of miRNA toolkit in HEK293 cells. (A) miRNA sensor plasmid to monitor the miRNA activity. The strategy for construction of AAV plasmids expressing functional pri-miRNA fragments (B) and miRNA inhibitors, TuD RNAs (C). Cross validation of miRNA expression and inhibition plasmids (D). Top at (D) shows increasing amounts of a pri-miRNA producing vector inhibits expression of a miRNA sensor plasmid in HEK293 cells. The bottom shows the de-repression from anti-miR TuDs in a dose response manner when we fix the amount of pri-miR plasmids. ITR, inverted terminal repeat; ΔITR, mutated ITR; CB, chicken β-actin promoter with CMV enhancer; U6, U6 promoter; PA, poly (A); pre-miR, precursor miRNA; Fluc, Firefly luciferase; Rluc, Renilla luciferase; β-Gal, β-galactosidase; 3 × miR-xT, 3 miRNA perfect target sites.