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UTSMC s David Corey Discusses RNAi and Getting It to Work In Vivo

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At A Glance

Name: David Corey

Position: Professor, University of Texas, Southwestern Medical Center

Background: Associate professor, UTSMC — 1998-2003; Assistant professor, UTSMC — 1992-1998; Postdoc, University of California, San Francisco — 1990-1992; PhD, chemistry, University of California, Berkeley — 1990; BA, chemistry, Harvard University — 1985

After jumping from one coast to the other in pursuit of his post-graduate degree, David Corey has settled down somewhere in between: the University of Texas Southwestern Medical Center. Recently, Corey spoke to RNAi News about his work.

How did you get involved with RNA interference?

I’ve been interested for many years in the recognition properties of chemically modified oligonucleotides, starting with regular DNA that’s modified with peptides or proteins. We’ve done a lot of work with peptide nucleic acids, a lot of work with locked nucleic acids. In my view, the ultimate recognition question [is]: What goes on inside a cell?

We became aware of how difficult it is to get good results inside a cell, so when RNA interference came along, we were very excited to start using it as a new approach for recognizing messenger RNA inside a cell and finding out how chemical modifications might improve that.

What’s some of the history you have working with the technology?

People here at UT Southwestern had been excited about [RNAi] for mammalian studies even before [Thomas] Tuschl’s paper [in Nature] came out [in 2001]. So when that did come out and we had road map of how to use it, me and another colleague, Mike White, started to use it. Mike in particular showed that it could efficiently knock down human caveolin.

That was working well, so we [in my lab] decided to take that as our model system. We started knocking down human caveolin, too, and putting modifications into the RNA to see how many modifications it would tolerate.

What did you find?

We found that it could tolerate complete substitution with phosphothioate linkages — if you’re talking about chemical modification to improve in vivo efficacy, that’s the first one that would come to most people’s minds. We saw that it could tolerate modification with locked nucleic acids, even though those greatly stabilize the duplex to the point where the melting temperature gets above 90 degrees [and] it still works quite well. We saw that it could tolerate 2’ fluro modifications.

That was a suggestion that there were wide opportunities for manipulating the properties of double-stranded RNA to improve its properties. Now, that improvement isn’t so necessary for uses in cell culture, but it’s going to be critical for transitioning to in vivo applications.

Where does that lead us to what your work is currently?

Of course, we continue to use siRNA as a tool that we use routinely when we want to investigate the function of genes, and we’re looking at using siRNAs and PNAs in tandem to amplify the amount of information you can get from cell culture studies.

As far as in vivo investigations are concerned: Earlier this year we concluded a bio-distribution study looking [at] where siRNA goes when it’s injected into mice. We found, as would be predicted from the literature for antisense oligonucleotides, most of it goes to the liver and kidneys. Also, [we found that] the siRNA is not nearly as stable in animal serum as it is in cell culture media.

Both of those things reinforced the idea that there’s going to have to be improvements in the chemical properties of double-stranded RNA to optimize what we see in animals and to make it [the] kind of robust technique that it is in cell culture.

What are some of the big challenges to making the transition to in vivo?

I think the big challenge is just that a laboratory interested in that area has to be dedicated to doing a lot of experiments, which means going through a lot of experimental animals and having access to diverse chemistries. Both of those get to be pretty expensive, and that’s probably best left to some of the companies that are working in this area — and there are some very good people, so I’m confident they can do it.

[As for] the particular take that we’re looking at: We have a long-standing interest in locked nucleic acids — that’s where there’s a linkage between the 2’ hydroxyl and the 4’ carbon. It allows much higher melting temperatures, so it’s a good way to stabilize a duplex through a minimal chemical change. We’re interested in asking the question: What happens when you put locked nucleic acids [into siRNAs] and is that going to improve efficacy in animals?

That’s the piece of the picture we’re going to be trying to look at.

Have you started that work?

We’re working with Proligo on that. Where we are now is we’ve gotten a series of unmodified RNAs. We’re going to test those first, see if we can get our foot in the door with something that works detectably, and then, once we have that, we’ll go in and make some locked-nucleic-acid derivatives and see if we can get it to work better.

So the big benefit of these locked nucleic acids is the increased stabilization. What are some of the problems you anticipate running into?

I don’t really anticipate any problems that couldn’t be solved by doing enough experiments. It’s just a question of how many experiments have to be done, because these aren’t going to be simple — you’re going to have to inject a significant amount of material into an animal, you’re going to have to harvest the tissues, you’re going to have to analyze them.

These experiments are going to be expensive, so that’s why we’re focusing only on one small aspect. As for problems, I would anticipate these things will be non-toxic [and] we already know they’re effective in cell culture. It’s just a question of having a bit of luck.

Delivery is first when you ask people about challenges to doing RNAi in humans. Is that something you’re looking at as well?

Well, stabilization is part of delivery: The longer it’s able to stay around intact, the more chance it has of getting to where it’s going to go.

Delivery is a problem, but I think that if siRNA is going to succeed or be an improvement on antisense, it’s going to be because it turns out to be more potent. If you get something that’s 10- or 100-fold more potent than an antisense oligonucleotide, then you need to get that much less into tissues and the delivery problem becomes less. I think that’s what people in the field should be hoping for. I could imagine modifying siRNAs with peptides or other molecules to enhance delivery or direct delivery to specific organs, and I’m sure people are going to be trying to do that. But that makes [the siRNAs] more complicated [and] they’re already twice the size of an antisense oligonucleotide. I think that if we’re talking about therapeutics in the near term for systemic administration, the molecules are going to have to stay relatively simple and easy to synthesize.

All that said, what is your take on the RNAi therapeutics field?

I think that what we need to be looking for from studies that are coming out is a demonstration of just how potent siRNA can be. You hear all sorts of numbers — sometimes it’s potent in the nanomolar range, other times people claim potencies in the picomolar range. I’d like to see a really good study demonstrating that yes, it is 10- or 100- or 1000-fold better than antisense oligonucleotides.

If you start seeing that, then I’d be very optimistic that people are going to be able to take the chemistries that have been developed for antisense oligonucleotides, optimize siRNA, and they’re going to work well.

You mentioned Proligo. Are you working with any other industry partners?

Not on the siRNA [front].

Aside from the work with stabilization and locked nucleic acids, you also do some work with RNAi determining the function of genes. What sort of projects are you doing on that side?

I’ll give you an example of work that we’re about to submit for publication: There’s a protein called major vault protein, which has been implicated in resistance to chemotherapeutic agents. We were able to knock that out and test that hypothesis very effectively. We’re also investigating the human progesterone receptor, and we did some work with human caveolin. We’ve also tried working with human telomerase. Of those four genes, we’ve only had problems with telomerase — the other three worked extremely well.

What were the problems with telomerase?

We couldn’t identify an siRNA that would work with what we would consider a promising potency. It’s a highly CG-rich gene; that may have been the problem.

What about working with RNAi down the road? Do you have any areas that you’d like to get into with the technology?

That’s hard to say. The field is moving so quickly [that] I can only look in the near term. What I would say is that we’re going to continue using it as a routine tool. It’s main function in my lab, though, might be to be a benchmark against which we measure all of our other efforts. We’re looking at peptide nucleic acids in depth, both as antisense agents and as anti-gene agents targeting chromosomes. SiRNA works so well that it’s really the standard by which competing techniques have to be judged.

What about other people’s work? Has there been anything you’ve seen as particularly important?

I haven’t had access to this paper yet, but I saw on Sciencexpress, where they put out articles that are pre-publication for Science, that there was one on using siRNA to silence at the transcription level; that’s going to be very interesting to read (see RNAi News, 8/6/2004).

[Also], I’m eagerly awaiting some of the data that I’ve seen in meetings where chemical modifications have been used to dramatically increase the potency of siRNA from low nanomolar to picomolar [levels]. If that passes peer review, again, I’m going to be very excited and optimistic about the future of [RNAi-based] therapeutics. If siRNA in cell culture can work in the low picomolar range, then I think it’s only a matter of time before it starts to find good in vivo applications.

Do you have any suspicions about claims people may have made about efficacy?

No, no real suspicions. It’s just that I’ve been in the antisense field for awhile, so until I can see data that’s peer reviewed with all the controls, I’ll withhold judgments. I’ll just hope that that data is out soon and is as good as it’s looked in presentations.

Do you feel people may have gotten burned by antisense?

Well, I think that the history of the antisense field is that you have to be careful interpreting data — if nothing else has been learned, that has been learned. The difference between good data and bad data comes out very clearly when you have them before you on a journal page, and if people in the siRNA field learn that — and I think they have since a lot of them are old antisense people — that’s going to speed up the field and save about five years of wasted effort.

 

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