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Mirus Dave Lewis on RNAi and Using the Technology at His Firm

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At A Glance

Name: Dave Lewis

Position: Senior scientist in charge of RNAi, Mirus

Background: Senior scientist, Mirus — 2000-2004; Postdoc, University of Wisconsin — 1995-2000; PhD, biochemistry, Michigan State University — 1993; BS, biochemistry/molecular biology, University of Wisconsin — 1981

After joining Mirus several years ago and working on the company’s initial gene knockdown experiments, Dave Lewis became the de facto head of the company’s RNAi program. With that role soon to become official, Lewis took time away from his work — and his newly arrived second child (it’s a boy) — to speak with RNAi News.

How did you first get involved with RNA interference?

When I was doing a postdoc, I was in Sean Carroll’s lab. Sean Carroll studies the evolution of development and generation of morphological diversity, things like that. We were working on a lot of strange organisms, trying to figure out what evolutionary changes occurred that would affect their development [and] lead to the different forms, mainly in insects.

The problem when you work in kind of non-model organisms is that there really are no tools that you can use to manipulate their genes in order to understand gene function. So, when I went to Sean’s lab, one of the things I was trying to do was develop tools that would allow us to manipulate gene expression in these non-model organisms.

About that time Andy Fire’s paper came out describing RNA interference and it really intrigued me, although at the time I thought it was maybe a pathway that only operated in something [like] C. elegans. So, I kind of put it on the back burner; in the meantime I was developing viral systems to try to infect butterfly wings, because we were studying butterfly wing color patterning as a model for morphological diversity. We were trying to manipulate those color patterns so I was making a lot of different viruses trying to find one that would infect butterflies and one that would allow gene mis-expression.

I was doing a lot of work there and then Richard Carthew’s paper came out describing the use of RNAi in Drosophila, and at that point we realized that RNAi might be more of a universal mechanism, which was great for us because, like I said before, there were no really good tools to actually manipulate gene expression in the species that we were looking at.

So, Richard Carthew’s paper came out, [and] I had a colleague that was working on beetles … he was studying their embryology and we felt we might try to use double-stranded RNA to manipulate their development and study Hox genes and how they functioned in beetles as compared to other insect species. We decided to just give it a shot: We made long double-stranded RNA and injected it into embryos, and it was like magic — we were getting phenotypes that directly mirrored some of the mutants that had been isolated by people working in that field.

It was relatively simple to do: Just inject the double-stranded RNA, let these things develop, and look at the morphology of the larvae.

That’s kind of how I got started in RNAi. It was also working with morpholinos and trying to get antisense to work. Morpholinos work to some extent, but nothing was as powerful as RNAi.

When you came aboard at Mirus, RNAi wasn’t something that was going on.

This was before [Thomas] Tuschl’s paper came out … which was in 2001, I believe. Up to that point, we were thinking about RNA interference, but people had only used long double-stranded RNA to induce it and, as you know, in mammalian cells that can induce the interferon response, so you get a lot of non-specific effects, which really would cloud any interpretation of experimental results on gene function. So, I kind of had [RNAi] in the back of my mind and, when I got to Mirus, I was actually working on using morpholinos and trying to deliver those to mice. We were having some success there and then the story about the small interfering RNAs came out. So, we pretty rapidly shifted from using antisense to using RNAs. Using techniques like tail vein injection, we were able to deliver it in mice and get an RNAi effect.

You came on as a senior scientist. At this point, you’re actually heading up Mirus’ RNAi efforts.

That’s right.

When did that happen? When did you take on that role?

It’s kind of by default. When I came to Mirus, it was strictly a gene therapy company and our main focus was to express genes — Factor VIII, Factor IX, things like that for hemophilia, [and] other genes, too. One of them was EPO for treating severe anemia. That was really my project, in addition to using the morpholinos as a kind of a side project.

So, when siRNA broke, I was really the only one here at Mirus working on that sort of approach: Gene knockdown, instead of gene expression. I just did a series of experiments in RNAi and got some good data, and right away started to apply for grants.

We were fortunate enough to get a couple of fairly large grants … At that point, the RNAi group here just got a lot bigger — right now we have seven or eight people working on it on the research side.

Another thing we do a Mirus, of course, is make research products for sale. So, early on, when we realized that delivery of siRNA was going to be the main problem in using this in mammalian cells, we decided to formulate a transfection reagent that people can use in vitro.

We were able to get a transfection agent on the market fairly early into the game, and so that has been very successful for us. Besides the grants, the products have provided an additional revenue stream to support the research.

So when was it that you officially moved to the position of head of the RNAi group?

It really hasn’t been made official yet. It’s imminent. But at Mirus, we don’t like to have a vertical management style; we like to have a team atmosphere. So, [the position] has been kind of by default, since I’ve gotten these grants that put this team together in RNAi.

These research projects — could you talk a little bit about what you’re working on now?

The focus at Mirus has always been delivery — that’s the key issue whether you want to do gene therapy or gene knock down with RNAi. And Mirus has always focused on trying to develop delivery technologies that are based on non-viral systems.

So, really that is what our focus is. We’re trying to put together formulations that we can inject into the vasculature that will deliver the gene or the siRNA to cells [and] to different tissues, [such as] internal organs.

Is the goal, then, to develop these technologies and then partner up with an RNAi therapeutics firm?

Yeah. That’s our strategy now, because we really feel that siRNAs are really not going to be a drug until delivery has been solved. Since that’s where our expertise lies, that’s the route we’re taking.

What sorts of tissues and areas are you looking at?

Right now, we’re looking mainly at delivery to the liver and to skeletal muscle.

Liver disease is easy to figure out — hepatitis, for example — but what sort of indications would fall under skeletal muscle?

For the skeletal muscle, that’s more probably on the gene therapy front. There aren’t a lot of targets that you’d want to knock down in skeletal muscle, although there are a few, like some of the dominant active muscular dystrophies.

For target validation purposes, and for just looking at how genes function in a certain tissue, really there are a lot of different targets you can think of in the muscle, although liver is the whole key to metabolism. There are a number of existing drug targets there, and there are a number of drug targets that are yet to be discovered.

Probably the most emphasis at Mirus, for RNAi, has been put on targeting the liver.

At this point, have there been any collaborations signed with RNAi drug companies?

We’re working with pharma, but I’m not in a position to disclose exactly who we’re working with.

Pharma in the RNAi therapeutics area?

This would be big pharma.

Are these big pharma partners looking at RNAi as a therapeutic modality?

Definitely. I think [just] about every pharmaceutical company is looking at that.

In terms of where Mirus is going in the RNAi arena … where do you see it going — sticking to the delivery side?

I think once delivery is solved, and delivery won’t be solved all at once — delivery is a complicated issue just because different organs require different strategies for delivery — but once that is achieved, then really you open up a whole host of different options. I think that, at that point, we’re definitely going to look into either partnering with pharma, or going out on our own and licensing in targets, and go down the therapeutic road.

But really, it’s such an early age now. RNAi in mammals is just barely three years old now, and to think that therapeutics is around the corner is, I think, a bit premature.

What’s a reasonable timeframe for you on RNAi therapeutics?

Well, it depends on delivery.

Would you say delivery to the liver …

I think that’s within reach.

So where would you target something like that?

In terms of a timeline — it could be next week, it could be next year.

Next week?

We’re using two or three different strategies for delivery to the liver and a couple of those look very promising right now. We’re following up with that, and as I’m talking to you we’re trying to do some proof-of-principle experiments.

Can you touch on the different approaches in as general or specific terms as you feel comfortable with?

One approach is to look at complexes. There are siRNAs complexed with a polycation polymer that is positively charged. Then, [we modify] that polycation in a way that will make it stable, so that when you inject it into the bloodstream, it will reach the tissue and be internalized by the cells.

Another approach is making complexes in a different way — not so much charge-charge interaction. Some of that looks pretty promising, but I can’t really go into any more detail than that.

A third approach is covalently modifying the siRNA to attach molecules that will aid in not only liver delivery but [in] liver targeting specifically.

One of the things we really specialize in is making complexes labile. We have good chemistry that allows us to really make these formulations or complexes labile so that once the siRNA gets into the cell, the delivery agent will fall away from the siRNA and allow the siRNA to be biologically active.

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