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Northwestern s Richard Carthew On RNAi and Where It s Headed

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At A Glance

Name: Richard Carthew

Position: Professor of molecular biology and cell biology, Northwestern University

Background: Associate professor, biological sciences, University of Pittsburgh — 1992-2001; Postdoctoral fellow, University of California, Berkeley — 1987-1992; PhD, biology, MIT — 1987; BS, biology, Queens University — 1978

Richard Carthew currently heads up his own lab at Northwestern University, where he pursues his interest in the molecular signals that control cell differentiation and morphogenesis during development. Through his work with Drosophila, he determined that RNA interference operates in species other than nematodes and incorporated the mechanism into his research. Recently, Carthew took time to speak with RNAi News about his efforts.

How did you become involved with RNA interference?

My interest in it began when I was attending a meeting of Pew scholars. … I was awarded that in the mid-90’s and we have an annual meeting [where] the fellows get together and present each others results. One of the fellow scholars in my class was Craig Mello, so in 1997 Craig discussed with us his observations concerning RNAi. The precise mechanisms were completely unknown — it was all phenomenology — but it certainly piqued my interest and the interest of our fellow scholars because it was such an amazing phenomenon.

That sort of planted a seed in my head that there was this thing out there that was quite mysterious but nevertheless very powerful. But it had only been observed in nematodes, in C. elegans.

Then, at the end of 1997, I phoned Andrew Fire, who is a friend of mine … about some unrelated matter and I asked him what was up. He told me about his discovery that double-stranded RNA would trigger the RNAi effect in worms. Because of the seed that had been planted, I knew a lot about how it worked in terms of the phenomenology, but it sort of crystallized in my head [that] this was truly a remarkable phenomenon and worthy of study. So, I asked [Fire] if anyone else had determined whether [RNAi] existed in other species, because in the back of my mind I was wondering [if] this was just a freak of nature, if you will. It had already been known that RNA metabolism in C. elegans is quite unusual — there’re things that go on in terms of RNA in C. elegans that you just don’t see very often in other species. And so, it was possible that this was simply a species-specific phenomenon.

Andrew hadn’t heard of anyone else seeing if double-stranded RNA could trigger silencing in other species, so I thought we’d give it a try. I immediately got a technician to perform experiments in Drosophila, essentially taking a known gene, which we had a mutant phenotype for, and trying to see if double-stranded RNA against that gene would phenocopy the mutant phenotype. Within a month, we had positive results.

We just basically flew with it from there.

Your working on, and have a government grant supporting, a project to discover the molecular mechanisms of RNAi. Can you talk a little bit about this project?

The project is essentially to discover genes that are important in the RNAi process using Drosophila as a discovery tool — essentially, create mutations in which RNAi is defective, impaired, or actually performs better than normal. Then, [our lab will] determine exactly how those genes function within Drosophila to affect RNAi.

It takes advantage of essentially a system in which we have the flies trigger gene silencing automatically — they express a transgene that synthesizes double-stranded RNA within themselves. So, we don’t have to add the double-stranded RNA or siRNA triggers to the animals — they basically do it themselves. That produces a nice, uniform silencing, and the target gene essentially gives a very easy readout, which is the pigmentation of the eye. What we then look for are mutations which change the pigmentation of the eye back to normal or actually make it even stronger.

That’s the nature of the screen. We’ve basically mutagenized roughly two-thirds of the genome and we’ve discovered quite a number of interesting genes, including what you might expect to pull out of the screen — for example Dicer genes.

Are there any specific genes you can talk about?

No.

When do you expect data from the effort to be made available?

We have two publications that are provisionally accepted in Cell, [but] they’re not accepted yet. That’s what I’ve been working on today — essentially to send off the revised manuscripts. Those describe the discovery, through this genetic screen, of Dicer mutations. We’ve come across some rather unusual finds with respect to Dicer that were unanticipated.

Are there any other projects you have ongoing that use RNA interference, or something down the road that you’d like to get involved with?

Of course, there are other genes coming out of the screen that we’re in the process of cloning and characterizing, but those aren’t ready for publication yet.

We’re interested in microRNAs, because there are … at least 100 of these in Drosophila, and indications appear that they have a developmental function. My primary interest, actually, has always been animal development. So, we’ve performed a genetic screen to essentially create mutations in microRNAs and then see what sort of developmental phenotypes we can identify. That’s been quite successful; we’ve identified mutations in four different microRNA genes and those are giving us very nice phenotypes. We’re putting that into a story.

Then, the final thing, is exploring the possible connection between Fragile X syndrome and RNA interference. There were a couple of papers published in December of 2002, which essentially identified the Fragile X protein in Drosophila as being a component of the RNA-induced silencing complex RISC. Of course, the immediate question is: Does this have anything to do with the disease state itself? Is Fragile X Syndrome caused by some sort of misregulation of RNA interference that normally occurs during human development?

So, we’re using Drosophila as the model for that because there is a homolog for the Fragile X protein in Drosophila — there are mutations in that gene. We’re essentially using the genetic tool that we’ve created to explore that issue of: What is Fragile X gene in Drosophila? How is it related to RNA interference or the microRNA pathway?

Just to change gears a little bit … what do you see as some of the disadvantages of RNAi, or some of the hurdles the technology needs to overcome to really blossom?

I’ll speak first from the Drosophila point of view, which is that [RNAi] is not robust enough, as yet, to be able to get routinely complete knockdown of activity — that is, there’s no gene activity. So, we’ve never reached what is known as the null state of gene activity. Rather, [RNAi] knocks it down maybe 90 percent or 80 percent. In some situations, that just not good enough — we don’t see a phenotype that we can actually score.

The other problem is that [RNAi] is more difficult to do in terms of knockdowns if we want to study phenomena that are occurring after embryogenesis — that is, after the animal has actually been born, still undergoing its development, or, say, if we were interested in behavior in the adults. [For] those kinds of issues, we have to rely more on transgenic technologies.

Speaking generally, say for interest in therapeutic purposes in human beings or research purposes in mammals … I think the problem with RNA interference is just like Drosophila — that is, there is not enough amplification of the silencing effect to reproducibly achieve 100 percent knockdown.

From the therapeutic point of view, delivery of siRNAs into cells within a living organism is, of course, a huge hurdle. Then, once they’re actually delivered inside cells, how long can they last before you have to undergo treatment once more? From the therapeutic point of view, I think there are some very big technical challenges.

Would you be able to offer any sort of predictions on the technology, looking out five years?

Yeah. I am very optimistic actually, despite the challenges, that [RNAi molecules] are going to be used therapeutically. It will be possible, not necessarily within five years, but it will be possible to do whole mammal siRNA gene silencing. Right now, we’re limited at the level of tissue culture dissociated cells, and it works quite well in that state. It’s now become … the reverse genetic tool of choice.

But I think in the next five years it’s going to expand to the point where one will be able to do what we can do in Drosophila now, which is silence gene activity within a whole organism … in a mouse, for example, or other standard model organisms, perhaps. That may pave the way for being able to essentially study … the genetics of animals which are non-genetic — for example, rats, pigs, dogs, cats, the kinds of mammals that are sometimes very useful as disease paradigms that the mouse is not good for.

That will be, I think, really important in terms of medical research.