Inhibitors of miRNA function promise to accelerate the understanding of miRNA functions in mammals, especially in adults. Chemically modified oligonucleotide miRNA inhibitors are effective, but are currently expensive, require repeated dosing that risks long-term toxicity, and many tissues are not currently accessible to delivery of oligonucleotides.
Transcribed miRNA-binding RNAs provide an alternative to oligonucleotides. Their small size makes them readily incorporated into a variety of gene transfer vectors. Primate AAV-derived vectors represent attractive tools for this application because of their unique tissue tropism, high efficiency of transduction, stability of in vivo gene transfer, and low toxicity.
We compared several designs of miRNA antagonists in vitro, then used the most effective design, TuDs, in vivo to inhibit miR-122 and let-7 by incorporating TuD expression cassettes into scAAV9. A single administration of rAAV9 expressing a TuD RNA efficiently depleted the targeted miRNA () and increased the abundance of its endogenous target mRNAs ( and ), which in turn produced the predicted phenotypic change in metabolism ().
Our data suggest that, in mice, TuD RNAs inhibit their miRNA targets via the target-RNA directed tailing and trimming pathway19
( and ). This work, in addition to observations in flies and cultured cells, suggests that the pathway is widely conserved among animals. Targeted miRNA destruction triggered by TuDs was surprisingly sequence-specific for the nine distinct let-7 isoforms. Compared to let-7a
, for which the TuD was fully complementary, a single purine:purine mismatch between the TuD and let-7e
reduced miRNA degradation by >3-fold, and four mismatches (let-7i
) reduced miRNA destruction 8-fold. We are therefore optimistic that future studies will reveal the rules for designing TuD inhibitors that target individual miRNA isoforms that differ by just one or two nucleotides.
The GLuc sensor system described here provides a simple means to detect changes in specific miRNA function, such as those caused by miRNA inhibitors, in live adult mammals across time (). This system allows one to assess the activity of a specific miRNA in a cell line, tissue or organ, providing a quantitative measure of the effectiveness of a miRNA antagonist.
Retrospective profiling has linked aberrant miRNA expression to a variety of diseases, suggesting that miRNAs may provide new targets for therapy. Indeed, miR-122 inhibition by AMOs5–8
or scAAV-delivered TuD RNA () lowers both HDL and LDL. However, the current view that HDL protects against heart attack argues that therapy for dyslipidemia should lower LDL but raise HDL levels. Recently, miR-33 was identified as a repressor of HDL biogenesis; miR-33 inhibition raises serum HDL level18
. Perhaps simultaneous inhibition of miR-122 and miR-33 by a pair of TuD RNAs expressed from a single scAAV vector may achieve a more balanced and healthy cholesterol profile and provide long-lasting therapy for familial hypercholesterolemia.
Low miR-122 levels have been associated with hepatocellular carcinoma in rodents and humans26–28
, although no direct causal link has been established27,28
. Because AAV vector expression is stable for years in rodents and primates, animals treated with scAAV9 expressing anti-miR-122 should enable testing the safety of prolonged miR-122 inhibition in general and the increased risk of developing hepatocellular carcinoma in particular.