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Harvard, Yale Team Devises Method to Protect miRNA Targets, Sees Potential in Rx Field

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Researchers from Yale University and Harvard University have shown that they can use morpholinos to protect a microRNA target gene from suppression in order to evaluate specific miRNA-mRNA interactions in zebrafish.
 
“Thousands of miRNA-mRNA interactions have been predicted, but less than a dozen have been shown to have an in vivo function,” according to the researchers, whose study appeared in last week’s Science. “The sequence selectivity of … target protectors makes them excellent agents to disrupt specific miRNA-mRNA interactions.”
These oligos “not only uncover the physiological role of miRNA-mRNA interactions but also illustrate how miRNA phenotypes are a composite created by up-regulation of multiple targets,” they added.
 
In the Science paper, the investigators used morpholinos complementary to miRNA binding sites in target mRNAs, hypothesizing that the overlap might interfere with miRNA-mRNA interactions and protect the target mRNA from the miRNA.
 
They achieved specificity using morpholino sequences specific to the 3’ UTR of target mRNAs.
 
“Basically, the most important feature of these oligos is that in the absence of the microRNA [they] shouldn’t do anything to the mRNA,” Antonio Giraldez, an assistant professor of genetics at Yale and co-author of the Science paper, told RNAi News last week. “You don’t obtain an artificial gain of function because [the target protector] binds to and stabilizes the mRNA independently of the presence or absence of a functional microRNA in that cell.”
 
While Giraldez sees great potential for the target protector technology in basic research, he noted that the approach, if combined with other kinds of oligos, might also be used therapeutically.
 
“In principle, any other … stable oligo that binds specifically to RNA should be able to inhibit microRNA activity,” he said. “I could imagine that 2’ O-methyl oligos or antagomirs, which are usually used to bind a microRNA, might be used to bind the mRNA.”
 
“By blocking the interaction of specific miRNA-mRNA pairs using target protectors, the translation and stability of particular mRNAs could be increased and result in the suppression of hypomorphic mutations or the up-regulation of beneficial gene products such as tumor suppressors or peptide hormones,” the paper’s authors wrote.
 
In the field of siRNA-based therapeutics, meanwhile, target protectors could theoretically be used to help deal with off-target effects, Giraldez noted.
 
For example, “if you have an siRNA targeting a cystic fibrosis gene and it suddenly starts to target another gene [causing] an off-target effect, you might be able to protect the off-target [gene] and have a more specific effect of a particular therapeutic siRNA” using a target protector approach, he explained.
 
“At the moment, we haven’t actually pursued [this kind of research] but I think it is feasible,” he added. “We have some labs here [at Yale] with whom we … will probably develop that using 2’ O-methyl [oligos].”
 
Giraldez said he also hopes that an industry player might be interested in commercializing the technology, noting that Harvard’s technology-transfer office will handle out-licensing negotiations.
 
Squint Meets Lefty
 

“In principle, any other … stable oligo that binds specifically to RNA should be able to inhibit microRNA activity. I could imagine that 2’ O-methyl oligos or antagomirs, which are usually used to bind a microRNA, might be used to bind the mRNA.”

In their experiments, the Yale and Harvard team focused on the zebrafish miR-430 family, which is “highly expressed during early zebrafish development, targets hundreds of mRNAs, and is required for embryonic morphogenesis and clearance of maternal mRNAs,” according to the Science paper.
 
Previous work with miR-430 suggested that it plays a role in dampening and coordinating levels of the TGF-beta agonist squint and the TGF-beta antagonist lefty.
TGF-beta, or transforming growth factor-beta, is a class of proteins associated with various cellular functions including proliferation and differentiation.
 
The researchers introduced morpholinos complementary to the region of the miR-430 target sites in the squint and lefty 3’ UTRs into zebrafish embryos. They found that protecting squint led to enhanced Nodal signaling, while protecting lefty reduced Nodal signaling.
 
Simultaneous protection of both squint and lefty, or the absence of miR-430, resulted in an “imbalance and reduction in Nodal signaling,” they wrote in their paper.
 
“Our study of miR-430 and Nodal signaling … identifies a role for miRNAs as dampeners and balancers of agonist/antagonist pairs and reveals a novel regulatory layer of Nodal signaling,” the authors wrote. “More generally, our results reveal a regulatory interaction in which a repressor … dampens expression of both an agonist and an antagonist.” 
 
Importantly, the work also “introduces a novel method to test the role of specific miRNA-mRNA pairs in vivo,” they added.
 
Although morpholinos proved highly effective in the investigators’ experiments, “other antisense oligonucleotides and small molecules that bind to miRNA target sites or their vicinities are also likely to serve as target protectors.”

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