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Aaron Diamond AIDS Research Center s Daniel Boden on RNAi and HIV

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At A Glance

Name: Daniel Boden

Position: Research scientist, Aaron Diamond AIDS Research Center, a Rockefeller University affiliate

Background: Assistant professor, Brown Medical School — 2000-2001; Research scientist, Humboldt University — 2000; Postdoc, Aaron Diamond AIDS Research Center — 1997-2000; Resident fellow, Harvard Medical School — 1996-1997; MD, University of Duesseldorf — 1996; BA, biology, Duesseldorf College — 1985

After working as a postdoctoral fellow at the Aaron Diamond AIDS Research Center, where he first started applying RNA interference to his research, Daniel Boden left for Brown Medical School to continue his work.

There, he and his colleagues came to the conclusion that HIV’s ability to mutate might be just as much of a challenge for RNAi-based drugs as it is for small molecules. As such, they proposed an approach termed multiference, in which multiple genes are targeted by RNAi at once (see RNAi News, 10/24/2003).

Now, back at the Aaron Diamond AIDS Research Center since January 2004, he spoke with RNAi News to give an update on his work.

You work in the [Martin] Markowitz lab? Could you give an overview of the kind of research that goes on there?

Most of the work here is very much clinically related. They do lots of clinical studies which entail also basic research studies. For instance, they have right now a study involving infusions of antibodies against HIV-1 — they follow these patients and see how well the virus is suppressed by this antibody cocktail, how long the suppression lasts, and what makes the virus come back.

Basically, it’s all HIV because we are an AIDS research center. The other study going on is gut related — [data] has just been published in the Journal of Experimental Medicine. We have a gastroenterologist here who is doing studies to look at assorted gut lymphocytes, and comparing viral replication by HIV [using] RNA and DNA analyses. He found that in the guts of patients, there’s a much higher viral burden [in these cells] compared to the blood. This is very interesting and striking news, and actually another paper in the same journal has [had] similar results.

There are a lot of drug studies here, for example with a new protease inhibitor. [The work here] is very broad and would be too much to get into. Essentially it’s all HIV.

So where do you fit in? You came over from Brown [Medical School] …

I was here before, actually. I’m mostly HIV trained, and at Brown I set up with a colleague a retrovirology lab mostly focusing on HIV. But I started … working on RNA interference as a means to suppress HIV infection or replication … here [at The Aaron Diamond AIDS Research Center], I think in 1999, before the defining of siRNAs and all that — I tried to come up with big double-stranded RNA constructs. Then there was the discovery of the small interfering RNAs, and I switched to short hairpins, and from there it all started.

But it’s basically all HIV related. The other thing I’m working on is microbicides … as HIV-entry inhibitors [but] they are not RNAi-based.

Now that you’re back [at Rockefeller] can you give an update on where your work led at Brown and what you’re doing now?

In one paper [published in the Journal of Virology], we mentioned that maybe a means to have better or longer suppression of HIV replication/infection is using what we called the multiference approach. [Now, with funding from a recent NIH grant, I am] using a natural microRNA cluster present in C. elegans, which constitutively expresses mir35 to mir41 microRNAs, as a backbone to express anti-HIV and anti-CCR5 microRNAs/hairpins.

Why did you select this cluster?

There are human microRNA clusters, but first of all the microRNA pathway is extremely conserved among different species. I chose that one [from C. elegans] because it’s a rather small cluster — the whole cluster, including microRNAs, is about 700 base pairs. If you look at other clusters, they’re much, much bigger, so the intervening sequences between one microRNA and the others can be up to a couple of hundred base pairs or even kilo-base pairs. So [the one I chose] is a very compact cluster.

In C. elegans, there is one paper showing that all these microRNAs are equally well-expressed, and since it’s a very conserved pathway, I figured [that I should] try … to optimize it for human microRNA expression and target the HIV genome, [as well as] the CCR5 co-receptor.

Where are you now? Where is the work?

What I did first is check … if the foreign microRNAs [in the cluster] are equally well-processed and expressed. Now, I’m working to see [what happens if] you slightly alter … the microRNA backbone artificially in terms of altering the thermodynamics on the 3’ end. There are a couple of papers showing that if you make [alterations] on one end … you can facilitate the uptake of the antisense strand, which is your guiding strand and the one you want to have taken up by the RISC complex.

First, I started with the natural [backbone] … and introduced some bulges, some mismatches, to see what is the best sequence context. I’m also comparing that to classic hairpins constructs to see on a single microRNA-hairpin basis which is the most efficient sequence context.

I’m also … looking to see how the expression of, let’s say, the second microRNA impacts the first one, and vice versa — to see if they’re really equally well expressed.

I’m also working on a very potent CCR5 microRNA, because the hairpins out there haven’t been shown to be extremely potent; you want to reduce CCR5 expression as much as possible because HIV-1 uses it early in infection. You want to knock that out, so I’m also working on the optimization of a CCR5 knockout.

At what point do you expect to move to in vivo studies?

Once you finish the in vitro studies to see that [the microRNAs] are all expressed, and you’ve figured out good targets … and you have all that in your cassette, the next step would be to take an HIV-susceptible cell line — you start with cell lines because they’re easy to work with — and come up with stable transduced cells that express that cluster, and try to infect them [with HIV].

Basically you first want to make a cell that’s immune to HIV infection through that microRNA cluster. Once you have the cell lines, you do the same in primary cells — you would transduce these cells and challenge them with HIV. If that give you a cell with intracellular immunity, then the next step would be monkey studies.

Since you’re at Rockefeller and you are working with RNAi, you must know Tom Tuschl. Do you have some sort of working relationship with him?

We’re friends, basically. We’re not collaborating on anything, but of course I talk to him for his advice and opinion on certain things. As you know, Tom is not so much into virology; his research is on RNAi in general — very, very basic. I’m more looking into applications of microRNAs and optimization of these systems.

As things move along, [do you expect] to see some industry interest?

Well, it would be interesting to have [this approach] applied on patients with multi-drug resistant HIV viruses which are not treatable with available drugs.

It would be a gene therapy approach, and gene therapy is still full of caveats and not really … approved by the FDA, although they use it for some cancer studies, and maybe it would be a last option for patients who don’t have any option left in terms of medications.

Ideally, what we would have then is transducing stem cells with [an RNAi] construct, and these cells would then produce T-cell that would repopulate under the selection pressure of HIV. [Non-resistant T-cell] would die, and [transduced ones] would not. That would be an ideal scenario but is purely theoretical.

There are companies looking into that. Have you had any discussions with working with anybody in industry?

Not really. I know that John Rossi [at the Beckman Research Institute] is already doing animal studies with a construct that has a ribozyme and an siRNA. But he’s at a bone marrow transplant center, so he can do these studies more easily.

 

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