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Ambion s David Dorris Talks About His Company s RNAi Activities

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At A Glance

Name: David Dorris

Position: Director of RNAi technologies, Ambion

Background: Manager of custom RNA services, Ambion — 2002-2003; Section manager, Motorola Life Sciences — 2000-2002; Postdoc, Harvard Medical School — 1994-2000; PhD, molecular and cellular biology, University of Texas at Dallas — 1994; BS, zoology, University of Texas at Austin — 1989

Although his real interest with RNAi started only a few years ago, David Dorris has become head of RNAi technologies at one of the industry’s most prominent players. He recently spoke with RNAi News about Ambion and what the company is up to.

How did you get started with RNA interference?

I joined Ambion, and Ambion had a very strong program in RNA interference. That was actually the most important reason I joined Ambion — to join [a company using] RNAi technology.

So, prior to Ambion, you hadn’t worked with RNAi?

I had not worked with RNAi directly [before Ambion]. I read publications and saw that it was very exciting. A few papers started coming out, and then you noticed that all of a sudden a deluge of papers starts hitting and it’s really clear that it actually does work.

Like most people, I didn’t really believe that it was going to work for that many genes and then it did. It was so obvious that this is a revolution in science from the basic research side, and then also it has possible therapeutic potential. But from just the basic research side, it was just so compelling — it’s what I dreamed about as a postdoc; it would have made my life so much better.

So in terms of what’s going on at Ambion in terms of RNAi, can you give me an overview?

We have the broadest product line of any company out there, and that’s everything [including our] biggest selling [RNAi] product, which is obviously RNA oligos for siRNA — there’s no doubt that that’s the most common use [of this product], especially because it’s so easy to get into cells.

We really focused a lot on the design, and we’ve partnered with a company called Cenix Bioscience for the design algorithms to design siRNAs. We’re almost finished with the next-generation algorithm, which is really superior to the first algorithm. By the time this publishes, that might be out.

The design … that’s the second most important thing. The number one most important thing is to be able to make very high quality RNA oligonucleotides. So, we’ve put in an incredible amount of effort on that at Ambion. It’s much more difficult than DNA, especially because RNA, at least siRNAs, are being used directly in cells. That’s a big consideration that I think a lot of people forget about when they’re used to ordering DNA oligos. There’s a big difference — these RNAs are being used in cells, whereas well over 99 percent [of DNA oligos] are just used in in vitro reactions.

That’s that part. We’re also very interested in delivery of siRNAs. We’re also very interested in delivery of plasmid vectors that have short hairpin RNAs. We’re also doing a lot of vector experiments and a lot of vector development — we have a very broad product line for that.

Can you talk about the new algorithm and how it differs from the one you’re using now?

The big difference is that we can go back [to Ambion’s dataset]. We have, I think, the largest dataset of anyone out there to actually look at function. [It’s] a dataset that’s trustworthy, and it’s also a systematic look at siRNAs that have been designed using a previous algorithm.

We have the data, so we know whether or not the siRNAs work, and so it’s very easy to identify certain motifs that are very prevalent [in highly functional siRNAs] … and very poorly functional siRNAs. …

The number one most important thing is that we can now identify, with a very high accuracy, the very, very functional siRNAs. That really separates us, I think, from everyone else. What that means is that you have to put a dose of reality in there and say that for some genes, you’re not going to be able to identify the best sequence because you can’t change nature — you can only design the best you can for that gene, based on the sequence that is available.

So, for some genes, you might not be able to identify an siRNA that’s going to work at the sub-nanomolar range for an IC50. But on the other hand, we think that it still might be possible. We’re not positive we can do it — we think it’s possible, but there’s no guarantee because you can’t change the sequence you’re designing against.

The main thing is that we went back and used the existing dataset that I think is broader and stronger that anyone else’s. The most important thing is that it targets hundreds and hundreds of genes — we’re not looking at a small number of transcripts with hundreds of siRNAs per transcript, which gives you a very biased view.

Can you talk about what sort of things you look for in an ideal siRNA?

Sure. We had already identified — and maybe we should have published it — a differential melting temperature between the 5’ end and the 3’ end for the sense strand or antisense [strand].

Essentially, we had already confirmed that Phil Zamore’s paper was correct. He showed in vitro very nicely that you can get preferential loading of the RISC complex by having the melting temperature at the 5’ end of the antisense molecule being significantly lower than the melting temperature at the 3’ end of the antisense molecule. Our design algorithm has always taken that into account.

The next step was to go and look at the placement of specific bases within that sequence.

[It’s] pretty simple, [although] it’s more than looking at just specific bases, by the way. You also have to look at combinations, and that’s where it gets a little bit more difficult.

You guys at Ambion are also looking at different delivery methods. What sort of projects do you have ongoing in that area?

The number one thing that we’re working on is reagents and methods related to electroporation. We have an electroporation buffer that is actually going to hit the market … [May 10]. It’s called siPort electroporation buffer. That’s a product we developed because we’ve found that electroporation is a very, very good method for getting all kinds of nucleic acids into primary cells and lots of cell lines that are of hematopoietic origin [and] that are very difficult to transfect with lipids.

We found that [electroporation] was very good, [but] on the other hand we found an incredibly high rate of cell death and a very significant amount of toxicity. That really made us question whether or not the method was useful — whatever population of cell you have, even if they’ve taken up some nucleic acid, you really question, if you’ve lost 50-60 percent of the cells, if the remaining cells resemble the initial cells. How injured are they? How stressed out are they?

So, we had to develop a proprietary buffer for increasing the cell viability during electroporation, and it’s actually been startling how well it’s worked. For many cell lines, we … get great delivery efficiency so that it’s well over 80 percent delivery, and at the same time we’re getting viability that’s well over 90 percent.

That’s for electroporation, and obviously we’re working on other things within electroporation that I can’t mention. The other thing we’re doing is looking at delivery using standard lipids and we’re very interested in that.

We’re testing multiple lipids and … we have some in the pipeline. It’s going to be happening very soon that we have a new lipid coming out that’s going to be quite good — much lower toxicity.

Will this extend into the RNAi therapeutics area at any time, or is that something you’re not looking at?

The next products [coming out of Ambion] are not really geared towards the therapeutic angle.

‘We have a lot of very interesting research projects going on’ is my answer to that.

So you could envision a time when Ambion expands … beyond basic research?

The answer to that, from a very different angle, is that we have a fully operation GMP facility that makes oligonucleotides. So, we have a full oligonucleotide manufacturing facility that is under full cGMP guidelines. Right now, we’ve been making DNA oligonucletides for diagnostic use, but obviously we can expand if needed for making … active pharmaceutical ingredients.

Would you have any comment on the RNAi therapeutics field? Do you have any sense of … how successful you think it’s going to be?

As I mentioned, we did start a GMP oligonucleotide facility and right now we’ve made well over 100 DNA sequences under full GMP procedures, which is actually quite a feat. We’re obviously looking at that market from the therapeutic point of view — that these companies don’t have GMP manufacturing capabilities. So, we’re ready for that if it comes. In that way, we’re positioning ourselves to be a player in that field if the market does explode.

Most of the technical issues, I think, will be overcome. Stability will be overcome in the next few years, so that people will be able to identify either very stable siRNAs or siRNAs that aren’t stable, if that’s what you want. …

The other thing is delivery [and] it’s just a matter of how many dollars people are going to put into it. There’re incredibly smart people working on it and a lot of compelling preliminary data that’s being shown and discussed.

Is this a contract manufacturing [position Ambion’s taking]?

Yeah, this will be contract manufacturing. Also, whenever we develop something here, we’re always trying to think if it has an application to therapeutics. So, obviously, there’re always possibilities.

Can you comment on what you see as the biggest challenges facing Ambion, not just from a technologic standpoint, but also from a business standpoint?

It’s definitely getting the word out to everyone — that’s for every business. Making people aware of your products is one of the hardest things. We can’t get them to log onto our website every day to look at every new product we have and that’s by far the most difficult thing to do.

The other thing is that … some of our competitors were in the market a little bit earlier than us, so we had to catch up there. We had to play catch up for a while, but now we’ve overcome that.

Who’s Ambion’s biggest competitor?

There’s an incredible amount of competition because the RNAi market and the RNA interference technology is so exciting and so important for the future of basic research, and possibly therapeutics. There are a lot of very formidable competitors.

Can you comment on the Benitec lawsuit (see RNAi News, 4/2/2004)?

No comment.

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