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TDI's Jerry De Zutter on Using RNAi to Find a Cure for ALS

Name: Jerry De Zutter
Position: Director, gene expression core, ALS Therapy Development Institute
Background: Postdoc, Wyeth Discovery Research — 2001-2005
PhD, molecular medicine, University of Massachusetts Medical School — 2001
Research associate, Genzyme — 1992-1994
BS, biological sciences, University of Massachusetts, Lowell — 1992

Amyotrophic lateral sclerosis is a neurological disorder characterized by the degeneration of motor neurons. There are two forms of the disease: familial, which is hereditary and accounts for an estimated 5 to 10 percent of all cases; and sporadic, which accounts for the remaining 90 to 95 percent and has no known causes.
The ALS Therapy Development Institute is a non-profit biotechnology company focused on discovering treatments for the disease. At TDI, Jerry De Zutter is responsible for running a gene expression-based research program to identify biomarkers and potential therapeutic targets for ALS.
This week, RNAi News spoke with De Zutter about his work and how the institute has incorporated RNAi into its research.
Let’s start with your role at TDI.
I was brought on to start a … molecular-based approach to understanding the disease. [The TDI had] done a lot of gross phenotypic analysis of drug-treated animals, specifically the SOD1 G93A [transgenic] mouse model [of ALS], and were looking at [things like] survival and hind limb function. That was pretty good in terms of carrying out efficacy studies and understanding if these mice are affected at all by whatever the treatment of choice was. But it really gave no insight into what could be happening molecularly.
I have experience in doing gene-expression analysis, so I was brought on to start up a … more focused approach to looking at expression events underlying the disease. Since I’ve gotten here, the whole program has really taken off. We [had] a sort of drug-screening function, we were filling that space in trying to understand the disease, but then we brought on a couple of people … and decided to begin more of a discovery research effort at the foundation … mainly based on using the mouse model.
That can be a number of different things, whether we’re looking at small molecules or protein therapeutics or siRNAs. We have a bunch of different things going on in trying to understand the disease in the mouse model. … We’ve made pretty good progress on using siRNAs in the mouse model, probably more progress in actually developing a pipeline for validating the siRNAs as being efficacious and doing what they’re supposed to do, and delivering them to the mouse model. We’re also attempting and having some success with putting these [RNAi oligos] in viral vectors.
Really the reason for doing this is not so much that we’re going to do a gene-therapy approach — that’s stretching it a bit in terms of how you could use RNAi in the mouse model — but more for validating targets that we feel could be important.
There are a couple of very interesting papers in the literature where people have used siRNAs either naked … or in viral vectors. The disease is amenable to this approach because these [molecules] can be delivered directly to the spinal cord. Initially, that [doesn’t] seem realistic because you can’t do that to a human, but in fact ALS patients many times have a port in their spinal cord through which things can be directly administered. We have set up in our lab … an intrathecal catheter [to] deliver whatever we want [to the mouse]. We’re doing that fairly regularly.
What we think is our advantage over a lot of other groups is that we’re doing this in a lot of mice. There are other studies published [detailing] similar approaches, but they’re not sufficiently powered with enough animals. … [But] we have enough mice … so that we can establish results that are really significant.
In addition to that, we’re implanting pumps so that we can have an osmotic infusion of the drug of choice, and we’re also doing this with siRNAs, over a longer period of time. So instead of having manual injections of a bolus through a catheter once a day, we have a continuous infusion daily … into the spinal cord for up to 28 days. The mice will also tolerate one pump change, so it could go out 56 days, and that really covers a good half or two-thirds of the lifespan of the G93A mouse model.
When you talk about using RNAi for target validation, with ALS are key targets for intervention not really apparent at this point?
There are a lot of different theories out there. People are starting to congregate in a couple of different camps, but there are a lot of ideas, things that people are very interested in: molecules that regulate the cell cycle, and that would affect apoptotic processes … redox reactions, oxidative stress, molecules that regulate the formation or breakdown of reactive oxygen species, heat-shock pathways, pathways that regulate protein folding. …There are a whole bunch of different camps as to what the molecular mechanisms are that underlie the disease.
We are trying to take a very broad view in terms of what we’re looking at. When we’re talking about RNAi and trying to interfere with the expression or function of some of these molecules, obviously we can’t take a global approach to knocking down all these molecules using siRNAs.
We’re going after things that are apoptotic pathways. … Of course the central molecule in most of the mouse models for ALS is SOD1, superoxide dismutase 1, and there are at least a hundred mutations to the molecule that have been correlated to the disease. It’s well established that it’s not a loss-of-function mutation — there is no problem with the normal activity of the molecule. In fact, the molecule is functioning normally in the mouse model. But what happens … is what’s called a gain of toxic property. Somehow, the mutation causes a gain of toxic function, and that’s where people ask, ‘What is the toxic property?’ Maybe its aggregation, maybe its oxidative stress — we really don’t know at this point.
It sounds like RNAi is very useful for target validation and figuring out the pathways that underlie the condition. But you sounded less enthusiastic about the possibilities of using RNAi as a therapeutic for ALS. Am I interpreting that right?
You are. I think it’s too new. …
We should clarify that there is a familial form of ALS, which is hereditary, and that actually [accounts] for a minority of the entire ALS population. The sporadic [form of the disease], where they’re not sure why it arises … are actually 90 percent of the cases.
When you’re talking about the familial form, a lot of people are honing in on that because they feel it is a model that is tractable to the research side of things. Indeed, after the initiation of the disease — and nobody is really going after the initiation of the disease [because] it’s too tall of an order at this point — it appears that the central mechanisms for neurodegeneration, whether it’s sporadic or familiar, are the same. So people are honing in the best they can on the familial form, specifically in the mouse model with the genetic defect, to try to understand that.
That being said, obviously SOD1 is a target. The only problem is that SOD1 is such a highly expressed and abundant molecule that to try to knock it down with RNAi is, again, a pretty tall order. That’s why you’ll see in the literature a couple of papers where people are putting siRNAs in lentiviral and viral constructs so you have a continuous production of the RNAi molecule to knock down the protein. Nevertheless, it’s [difficult] to knock down a molecule that is that abundant.
This would essentially fall under gene therapy, which is a tough sell right now. It’s gaining momentum again, but every time there is a setback … everything comes under intense scrutiny. With siRNAs, people don’t understand them that well, [and] the mechanisms of how it works have only recently been established.
It would be nice if [RNAi approaches] could turn out to be a magic bullet seeking out the appropriate molecule, knocking it down, and leading to an arrest of disease. But right now, I think we need to focus in on using it more as a validation tool.

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