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Novartis William Wishart Discusses RNAi at a Big Pharma

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At A Glance

Name: William Wishart

Position: Head of functional genomics/chronic pain project, functional genomics department of the Novartis Institutes for Biomedical Research

Background: Program head, Novartis neurogenetics program — 1995-1999; Senior scientist, Sandoz Pharmaceuticals — 1987-1995; Postdoc, molecular biology, Princeton University — 1884-1987; PhD, molecular microbiology, SUNY Stony Brook — 1984; BS, biochemistry, University of Connecticut — 1979

After finishing his postdoctoral work at Princeton, William Wishart entered the world of big pharma at Sandoz, a company that can be considered his home to this day, given that Novartis was created out of the 1996 merger of Sandoz and Ciba-Geigy. Working his way up through the company, he is now head of functional genomics for the company’s biomedical research institutes and focuses on drug target validation.

Wishart recently took time to speak with RNAi News about how RNAi is used by Novartis and where the company might be going with the technology.

I always start by asking how you first got involved with RNA interference.

That’s a long story, but to make a long story short: What happened was we worked on antisense for several years and we actually set up a system for doing antisense in vivo and were very excited about those results.

Actually, we published the work that we did on an ion channel [associated with pain signaling] called P2X3. There we showed that you could actually down regulate P2X3 in vivo and it gave a nice behavioral result.

About that time, that’s when RNAi became popular. The [antisense] system that we had [was] set up, and that included a mini-pump administration of oligo intrathecally. Everything had been worked out, and we literally just had to do some testing with RNAi versions of our antisense constructs to check to see if they worked in vitro first. But then, we were able to simple administer them with the same pump, the same system, in vivo and it just worked beautifully.

Basically, it was taking advantage of the system we had spent years setting up and, in the end, the work went very quickly.

What was the purpose behind using antisense? Was it strictly target validation or were you looking at therapeutic applications?

The focus was really validation, and that’s primar- ily the focus of all the work that we’re doing with oligonucleotides, previously with antisense [and now] with siRNA.

Maybe I can make one point: One thing people don’t often realize is that if you want to validate a target in the world of pharmacology, many people consider compounds the best means for doing that. There, I have to agree; if you have the specific compound, then that’s a great tool for validation, and of course it provides you with a good lead for developing a drug later on.

But, in many cases, you’re working with a family of receptors. When you’re in a situation where you have several receptors, then often there are not compounds available that are specific. So even in that case, where there are some compounds available, still RNAi can be an advantage because of the fact that you can go in and specifically knock down members within a family [of receptors].

That is an advantage of RNAi over the use of compounds.

At Novartis, is the transition from antisense to RNAi complete?

I would say so, yeah. One of the final points that drove us in that direction was simply the results of toxicity work that was done.

Occasionally we would see problems with antisense, and I have to admit that with RNAi, with siRNA, we haven’t seen any sign of toxicity — we haven’t lost any animals, we haven’t seen any paralysis, not even any sign of discomfort. I think that’s probably based on the fact that it’s a natural mechanism that we’re using, whereas the antisense is more of a synthetic type of compound.

Can you give an overview of how Novartis uses RNAi and how the experiments are designed?

For example, with the P2X3 work … we went through a phase where we tested the oligos in vitro. There, because of the expense of the in vivo work, we tried to characterize the oligos in vitro as much as possible.

So, we start with a selection of oligos. We then do screening for RNA inhibition with RT-PCR. We also screen at the level of protein[s], and if possible, we also try to get some functional studies done in vitro, as well.

With P2X3, it was relatively straightforward because we had a FLIPR type of assay. Initially we had electrophysiology, where we could actually look for effects on function. But we also had a FLIPR assay, which allowed us to do a relatively through analysis on the siRNA.

In other words, we went through the whole thing: RNA reduction, protein, and function. So, we were very confident that if the oligo hit its target in vitro, it would knock down the target in vivo, as well.

You can’t always do that with every target you have, but in general, the more information you can collect about your siRNA in vitro before you go in vivo, the more confident you can be about the in vivo results.

At this point, how much is RNAi part of Novartis’ target validation activities? Is it across the board?

Well, it’s being used in practically every department now. In functional genomics, we’re kind of sitting in the center, and because of the technologies we have, we can apply them to practically every disease area. So we collaborate with practically every disease area.

SiRNA is used as a tool, and it’s used when there aren’t specific compounds available. Occasionally, you run across targets where there are compounds in the literature or there are compounds available in our compound libraries that we can use — that’s why I said before that most people would agree that if you have a compound, a specific compound, that’s obviously the best place to start.

In the end, we sell drugs, we sell pharmaceuticals. So often we’re thinking in terms of validating targets, and once they’re validated, then to set up a full-blown medicinal chemistry program and develop a good drug from that.

So, if you have already a small molecular-weight compound that you can use as a starting point, then that’s a great thing. But normally, that’s not the case, especially with the newer targets coming out; often these are rela- tively unknown, or unannotated, or unidentified targets.

That’s another advantage of the oligonucleotide approach. Even if you don’t know what the target actually does, you can still go in and take a look in vitro or even in vivo to see if it has an effect on the disease. Once you have that, once you see that you can affect the disease state — and it doesn’t have to be 100 percent, if you can decrease pain 50 percent or significantly — then you know that that target is playing a role. The chemists can go in very confidently and attack that target knowing that there’s a good chance that that it will work in the clinic. In that sense, [RNAi] can also save us a lot of money, by allowing us to kill unimportant targets early and to focus on targets that have a direct effect on the disease state. …

[So] if you don’t have a compound, or you’re working with a gene family that might be difficult to dissect with a compound, then the siRNA is a very powerful and important tool.

How does Novartis go about designing its siRNAs?

We have a general approach: We prepare many oligos and then screen through them. As I mentioned, we have a reporter assay where we can literally look across hundreds or thousands of oligos to check for activity.

But, my feeling is that in the future, once we have gathered enough information, we can start setting up neural network-type approaches. I expect that there will be rules that will come out that can be used for automated selection.

Neural network? How do you mean?

What I’m talking about is biological information across maybe thousands of oligos. If you can see which ones are active and which ones aren’t, then it’s possible to think of perhaps feeding a neural network and developing a tool for doing that.

But at the moment, the primary means is simply selection.

Where is Novartis going with its RNAi activities? Are there plans in the works for a broader approach to using the technology, given Novartis’ recent deal with Qiagen for a human genome-wide siRNA library?

The general idea is to set up a situation where you can actually look at any target that comes out with a comprehensive set of siRNA reagents. Once that’s available, I’ll probably be out of a job, so then the robots can take over.

In terms of other uses for RNAi, is Novartis looking at the therapeutic applications of the technology?

It’s something that we’re obviously looking at. People are talking about that quite a bit, and my feeling is that, based on the results we have in vivo in animal models, that you can certainly think in terms of some day applying that therapeutically.

We’re applying a compound that’s apparently non-toxic, and we’re having a direct effect on a disease phenotype, so of course it’s a natural or logical next step to think in terms of some day applying that to humans. It’s being discussed, but it’s really to early to make any direct comment on our thoughts or plans there.

It’s just too early. But certainly the possibility exists, and we should [consider it] for the sake of the patients. And there are many patients in the field of pain who certainly could perhaps be helped by these technologies, or in spinal cord regeneration.

But at the moment, I can’t make any specific comments.

I’m getting the sense that at this point it’s just discussions [at Novartis], there isn’t an actual preclinical program underway examining RNAi as a therapeutic.

Right. I think that would be the best way to put it. We’re discussing possibilities … and I can imagine some day that it might be applied in the clinic. But I also have to admit that it’s still very expensive and that the primary use of [RNAi] right now is in target validation and proof of concept.

Do you have plans to take time off for the holidays coming up?

Yeah, briefly. Our boss said that our slides for our next presentations for the departmental meetings have to be in on January 5.

So, it’s going to be a busy holiday.

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