Long-term, efficient inhibition of microRNA function in mice using rAAV vectors

Abstract

Understanding the function of individual microRNA (miRNA) species in mice would require the production of hundreds of loss-of-function strains. To accelerate analysis of miRNA biology in mammals, we combined recombinant adeno-associated virus (rAAV) vectors with miRNA `Tough Decoys' (TuDs) to inhibit specific miRNAs. Intravenous injection of rAAV9 expressing anti-miR-122 or anti-let-7 TuD depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti-miR-122—but not anti-let-7—TuD reduced serum cholesterol by >30% for 25 weeks in wild-type mice. High throughput sequencing of liver miRNAs from the treated mice confirmed that the targeted miRNAs were depleted and revealed that TuD RNAs induce miRNA tailing and trimming in vivo. rAAV-mediated miRNA inhibition thus provides a simple way to study miRNA function in adult mammals and a potential therapy for dyslipidemia and other diseases caused by miRNA deregulation.

The use of high-throughput sequencing methods for plant microRNA research

“…98 Notably, a novel kind of sponges called anti-miRs is becoming a strong candidate for miRNA-based therapeutics. 43,[99][100][101][102][103][104][105][106][107] The anti-miRs are chemically modified oligonucleotides developed to modulate the activities of specific miRNAs. A recent study carried out by Hogan and his colleagues 108 demonstrated that the anti-miR physically associated with the AGO protein complex in the context of the cognate target miRNA both in vitro and in vivo.…”

Strategies to deliver microRNAs as potential therapeutics in the treatment of cardiovascular pathology

“…Such adaptations of the miRNA sponge concept allow longer-term inhibition of miRNAs than can be achieved by transient transfection of RNA polymerase II sponge reporter vectors or modified RNA oligonucleotides. Xie et al (2012) combined recombinant adeno-associated virus (rAAV) vectors with miRNA TuDs to inhibit specific miRNAs. Injection of a rAAV9 construct expressing anti-miR-122 or anti-let-7 TuDs resulted in long-term depletion of the corresponding miRNA and increased expression of its mRNA targets.…”

RNA accessibility impacts potency of Tough Decoy microRNA inhibitors

“…Studies have also shown that RNA polymerase II promoters can be used to efficiently express TuD inhibitors within the cell [24,25]. It has been shown that adeno-associated virus-delivered TuDs induced long-term miRNA inhibition in mice [27,28]. TuDs and sponges are both more effective when the miRNA binding sites are non-complementary to the target miRNA at position 10–11, presumably because Ago2-dependent cleavage of the TuD is prevented [23,25,29].…”

“…TuDs and sponges are both more effective when the miRNA binding sites are non-complementary to the target miRNA at position 10–11, presumably because Ago2-dependent cleavage of the TuD is prevented [23,25,29]. Whether TuD activity is mediated by miRNA sequestration alone, or also by miRNA degradation remains unclear, since several studies have reported conflicting results [20,23,27]. In comparative studies, TuDs proved more effective miRNA inhibitors than sponges [23,27].…”

“…Whether TuD activity is mediated by miRNA sequestration alone, or also by miRNA degradation remains unclear, since several studies have reported conflicting results [20,23,27]. In comparative studies, TuDs proved more effective miRNA inhibitors than sponges [23,27]. Recently, an AAV-based TuD has been successfully used to restore cardiac function in a murine heart failure model by targeting miR-25 [28].…”