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MIT's Phillip Sharp Discusses microRNA Sponges

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Name: Phillip Sharp
 
Position: Institute professor, biology, Massachusetts Institute of Technology
 
Background: Director, The McGovern Institute, MIT — 2000-2004
- Head, department of biology, MIT — 1991-1999
- Nobel laureate, physiology or medicine — 1993
- Director, Center for Cancer Research, MIT — 1985-1991
- Senior research investigator, Cold Spring Harbor Laboratory — 1972-1974
- Postdoc, Cold Spring Harbor Laboratory — 1971-1972
- PhD, chemistry, University of Illinois, Urbana — 1969
- BA, chemistry/mathematics, Union College — 1966
 

 
This month, Phillip Sharp and colleagues at the Massachusetts Institute of Technology published data on so-called microRNA sponges, which are transcripts expressed from strong promoters that contain multiple tandem binding sites to an miRNA of interest.
 
According to Sharp’s paper, which appears in Nature Methods, when vectors encoding the sponges are transiently transfected into cultured cells, the sponges derepress miRNA targets at least as strongly as chemically modified antisense oligos.
 
This week, RNAi News spoke with Sharp about the sponges and their potential applications.
 
Let’s start with an overview of the microRNA sponges and how they were constructed.
 
The premise of the microRNA sponge is a pretty straightforward idea. It’s a classical dominant negative where you transfect in vast excesses of a target and suppress the active component within the cell.
 
In this case, the vast excess is a messenger RNA-type structure that has many target sites for microRNAs. In theory and in practice, the microRNAs will bind to it and be sequestered from binding to [mRNA] that appears in the cytoplasm and therefore not suppress its normal target.
 
The neat thing about the technology as devised is you put a reporter with the sponge microRNA expressed at very high levels with many binding sites. When that reporter is expressed in the cell and the protein is made, you know you’ve inactivated the microRNA population to such an extent that it is no longer active and suppressing the expression of the genes.
The other thing that is nice about this particular sponge technology and approach to inhibiting microRNAs is that microRNA activity as a family of microRNAs is mediated through a common seed region — positions 2 through 8. All microRNAs with a common seed are thought to interact with a common set of target messages. By inhibiting through this seed region, you … inactivate all the microRNAs of that family, and that’s really what distinguishes this from using a specific oligonucleotide with an antisense technology in the same way.
 
Can the sponges be designed such that they go after specific microRNAs or sets of microRNAs?
 
They are designed to go after a specific set of microRNAs that have a common seed region and therefore are considered biologically to be of an overlapping family of microRNAs. That is really the important aspect.
 
You can use a 2’ O-methyl-modified RNA or other modified single-stranded [nucleic acids] to inactivate a specific microRNA. But to inactivate all of those of a common seed family with a common biological activity, it’s more complicated than using the sponge-type of approach with the specificity being for inactivating microRNAs with a common seed region.
 
And that was the driving force behind designing these sponges?
 
Yes, that was the driving force for it.
 
When you were examining the efficacy [of the sponges], what sort of levels of microRNA suppression were you seeing?
 
It depends on a particular cell and the target, but we could easily get, in a system where we knew the target and the microRNA, greater than 70 or 80 percent suppression of the activity, or even higher.
 
When we were looking at endogenous targets, meaning endogenous microRNAs interacting with their target messenger RNAs controlling proteins in those cells, the typical result … was a two-to-three-fold suppression of expression. We could basically repress more than half of that, but not completely. But we’re getting thinned out into the area where the precision of the measurement is important.
 
The work detailed in the paper was done in vitro, correct?
 
It was done in cells in culture, yes.
 
At this point, have these been tested in animals or is that work ongoing?
 
There is a paper cited in [our Nature Methods paper] that used the technology in an adenovirus vector. It is possible, probably likely, that one could design a transgenic expression vector where you could use [the sponge technology] in mouse models from a transgenic mode of expression.
 
The challenge there is to get a high enough level of expression. Certainly in some cases you’re going to be able to do it, but [using it] across any target gene will probably [require] some more perfection as a technology.
 
What do you see as the key research applications for this technology?
 
There is increasing interest in identifying all the targets of a given microRNA and microRNA family, [as well as] trying to determine the role that these microRNAs play in various biological transitions. I think this technology will be very valuable in doing that.
 
We know there are microRNAs that seem misregulated in cancer and important in developmental transitions and other things, and tools to identify the suppressor activity are going to be valuable in studying that biology.
 
What about therapeutic applications? Do you see potential there as well?
 
I don’t think therapeutic applications with the sponge technology as described are likely. It would require a gene therapy-type approach where you would use a virus to introduce the DNA vector that would be a sponge, and therefore you have the complications of the whole gene therapy thing.
 
In time, if the DNA delivery side of this to cells in vivo can be solved to be safe and efficacious, then there is a possibility this could lead to therapeutic uses directly. I think the more common use of it [will be] in mouse models or cell models for actually identifying the target of microRNAs in terms of their regulation and their biological activity.
 
And then an antagomir or locked nucleic acid approach …
 
Yeah. Then directly inhibiting the specific microRNA is more likely to be the approach used.
 
At this point, is more being done by your lab on these sponges or is it now out there and left to other researchers?
 
Well, it is out there and I’m sure other people will try it and use it and perfect it. And that’s great.
 
We’re likely to try the approach of doing a transgenic expression module. This was all thought up by the graduate student who is the first author on the paper, [Margaret Ebert], and she’s got 20 ideas for every one she can do.
 
Have you gotten any interest from industry on this technology?
 
We talked about this technology about a year ago or so at meetings, and we’ve had inquiries about it. But I don’t know the status [of those discussions] right now.

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