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Brigham & Women s Anna Krichevsky Discusses microRNAs and Brain Cancer

Anna Krichevsky
Brigham and Women's Hospital

At A Glance

Anna Krichevsky

Instructor, Brigham and Women's Hospital

Background: PhD, micro- and cell biology, Hebrew University, Jerusalem — 1999

BS, biology, Second Medical School at Moscow — 1991

As an undergraduate in Russia, Anna Krichevsky thought about pursuing a medical degree. However, she opted for a career as a researcher and eventually ended up as a postdoc in the lab of former Harvard Medical School professor Ken Kosik, where she developed a technique to deliver siRNAs to neurons in culture.

Now she has her own lab, and recently spoke to RNAi News about the direction she is headed.

Now that you've settled in with your own lab, maybe you could talk a little about what you're focusing on.

Our general interest is microRNAs and other non-coding RNA molecules. We are interested in microRNAs in several respects. The first is in brain tumors. We are also interested in the role of microRNAs in brain development and neuro-differentiation. But our first priority and real focus for right now, because we're still a very small lab with few people, is microRNA function in brain tumors.

You probably saw our recent paper in Cancer Research, co-authored by Jennifer Chan and Ken Kosik. We have used a microRNA chip, produced here in house, with probes for 181 specific microRNAs to profile microRNA expression in brain tumors. It seems that malignant brain tumors, glioblastomas, show a very distinct expression signature with specific microRNAs strongly over-expressed in most tumors. We can actually find a single microRNA that is extremely highly expressed in all glioblastoma cases — I'm talking about primary tumors — that we have tested. [This is also the case] in multiple glioblastoma cell lines that we have been testing. It's not just that this specific microRNA, called mir-21, is expressed at very high levels there, [but] it seems that inhibition of this specific microRNA in glioblastoma cell lines can cause apoptotic death of these cells. So basically we are really interested in pursuing [these] data.

What's the next step now that you've identified mir-21?

So the next step, once we saw that it was strongly over-expressed, was to inhibit its expression in these glioblastoma cell lines. And we do this using modified antisense oligonucleotides that basically block the function of this microRNA by preventing its incorporation into RISC. What we saw was that once this microRNA was blocked, and its level in glioblastoma cells becomes quite undetectable — very insignificant — these glioblastoma cells undergo apoptosis. These data suggest that mir-21 is more than a diagnostic marker: Its over-expression in glioblastoma may be important for development or maintenance of this tumor. We're really excited about this finding and are currently trying to understand exactly how is mir-21 is involved in preventing glioblastoma cell death. The idea is that mir-21 may contribute to the malignant phenotype by silencing expression of critical apoptosis-related genes.

I strongly believe that mir-21 may represent one of the first examples of small RNA regulators acting as micro-oncogenes.

The goal, then, is to try this out in vivo?

Absolutely. That would be the next step.

Timewise, where do you stand in terms of trying this thing out in animals — I presume mice, right?

Yeah. We're just starting these experiments so we don't have any data to talk about.

So you're actually doing the in vivo work now?

We're starting to. Of course the goal would be to inhibit mir-21 expression in tumor models and to see whether this has a similar effect on tumors in vivo as it had in glioblastoma cells in vitro.

These antisense oligos you use, what are they?

They are single-stranded, modified oligonucleotides. Some of them have been described by Tom Tuschl and Phil Zamore, and those are 2'-O-methylated oligoribonucleotides. Also, we were using so-called LNA oligos, and those are DNA oligos with incorporated locked nucleic acid nucleotides. They [both] seem to have very similar effects on microRNAs.

Are you going to try experimenting with both in vivo?

Yes, we probably will.

Have you done anything with short interfering RNAs or hairpins or anything like that?

Yes. We actually have tried several siRNAs to reduce the level of mir-21 precursor. It seems that they get some degree of effect, but it was not nearly as potent as an effect of 2'-O-methyl or LNA oligos. But we didn't really try systemically to find the best possible siRNA for reducing mir-21 levels. It would be very interesting work to do.

Do you get the sense that you'd be able to go forward with these antisense oligos in vivo? In terms of what people have done in animals and in clinical trials, a lot of times antisense oligos weren't as effective.

I should emphasize that we are talking here about a principally novel group of targets for drug development — microRNAs. At this stage, based on in vitro experiments, LNAs seem to be more potent miRNA inhibitors than small interfering RNAs. Nevertheless, we have collaborations with companies, and we definitely will try to test additional small interfering RNAs.

These oligos, do you create them yourself?

No, we purchase them from companies. Also, the whole topic is very new, so at this stage what we have running in our incubator is an experiment where we compare different LNA oligonucleotides from different companies with really minor differences in their structures. It seems that some minor differences could make big differences in their effect.

So who are you collaborating with?

We have several collaborations with biotech companies, including Exiqon, which produces the LNA oligos. We are testing their LNAs, which seem to be really good for in vitro purposes.

Anyone else?

We will do in vivo experiments in collaboration with other academic labs, including the lab of Peter Black here at Brigham. They have different tumor mice models of human glioblastoma. We also collaborate with Chris Sander and Debbie Marks from Memorial Sloan-Kettering Cancer Center on bioinformatics of miRNA. And, of course, we will continue our long-lasting collaboration with Ken Kosik, [who is now at the University of California, Santa Barbara].

The idea is that this could develop into a human therapeutic for brain tumors.

Of course, that is our dream.

Do you get the sense that these oligos themselves could be therapeutics, or do you see … small molecule intervention [as the more likely route]?

It's probably too early to talk about therapeutics because we don't have in vivo data yet. Our hope is that we'll be able to find … a modified antisense molecule, like LNA … that acts as a microRNA inhibitor, and by inhibiting the microRNA [it could] affect tumor growth.

Once you actually start getting in vivo data, how are you going to deal with the delivery issue?

It's very hard to make progress in developing a drug for brain tumors. Most malignant brain tumors like glioblasoma multiforme are absolutely incurable, and any small step toward finding a cure would be a great success.

We're planning to go step by step. The first step will be proof-of-principle to show that by inhibiting microRNAs, assuming the inhibitor is delivered in the right way, we can inhibit tumor growth. In other words, we want to test whether mir-21, and microRNA in general, can be a good drug target. Then, if it works on subcutaneous models of brain tumor, we can move toward targeting microRNAs in intracranial tumors. And, delivery will of course be a big issue here that will require a collaborative effort of academic and pharma labs.

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