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Q&A: Georgia Tech's Niren Murthy on Oral Delivery of siRNAs

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murthy3.jpgName:
Niren Murthy

Position:
Associate professor, biomedical engineering, Georgia Institute of Technology

Background:
• Assistant professor, biomedical engineering, Georgia Institute of Technology
• Postdoc, chemistry, University of California, Berkeley
• Research assistant, biomedical engineering, University of Washington
• PhD, bioengineering, University of Washington — 2001
• MS, bioengineering, University of Illinois at Chicago — 1996
• BA, political science, University of Redlands — 1992

Researchers from the Georgia Institute of Technology and Emory University this week published a report in Nature Materials demonstrating the ability of a novel nanoparticle to deliver siRNAs to sites of intestinal inflammation when administered orally to rodent models.

The siRNAs inhibited tumor necrosis factor-alpha, a pro-inflammatory cytokine implicated in a variety of inflammatory diseases, in the animals' colons and protected against ulcerative colitis, according to the paper.

Gene Silencing News spoke with the study's lead author, Niren Murthy, about the findings.

Let's start off with a bit about your research focus and what led you to start working on RNAi delivery.

Our laboratory focuses on developing new materials for drug delivery and molecular imaging. The molecular imaging projects in our lab have a big focus on imaging reactive oxygen species, so we have a very strong interest in trying to develop new ways of quantifying the amount of reactive oxygen species in tissues and [working on] in vivo imaging of reactive oxygen species.

We spend a lot of time looking at types of chemistries that will uniquely react with reactive oxygen species. There are a lot of different chemistries floating around in our lab, and one that is particularly interesting is a thioketal molecule. A thioketal is a very commonly used protecting group in organic chemistry because of its unique property of being stable to acid, stable to base, and only being cleaved under oxidative conditions.

So part of my lab is very interested in reactive oxygen species and what cleaves selectively in [them]. The other part of our lab focuses on drug delivery, particularly molecules too big to diffuse inside of cells, and siRNAs are great candidates there.

We had a lot of discussions and brainstorming, and one project that seemed particularly interesting was to see if we could make nanoparticles of thioketals and then [use them to] deliver [siRNAs] to treat diseases of intestinal inflammation.

The idea would be that you would … make a nanoparticle that could orally deliver siRNAs and protect them because of [the thioketal's] stability in the acidic environment of the stomach … [and] the very high enzymatic degradation capacity of the intestine. Also, only where there is reactive oxygen species would [the nanoparticle] be able to release the drug because of the intestinal inflammation and the reactive oxygen species link. The other thing is that the synthesis of a polythioketal is related to other molecules we made in our lab called polyketals.

So this project seemed feasible, and it's been about three years of work. It took about a year to make the particles and get comfortable with that. We had an excellent collaborator at Emory University named Didier Merlin who is an expert in inflammatory bowel disease, and with him we did a bunch of in vivo experiments.

Had you worked with RNAi before?

This is our second paper on RNAi. We had another one that was published in Nucleic Acids Research on using microparticles of polyketals to deliver siRNAs to liver macrophages.

Can you give an overview of the nanoparticles?

The nanoparticles are composed of a polymer called a polythioketal, [which is] … made by taking a dithiol and doing an acetal exchange reaction that gives us the polymer. The really nice thing about a polythioketal is that it is just incredibly hydrophobic. It is the most hydrophobic biodegradable material I've ever seen, and what's important about the hydrophobicity is … that it forms really nice particles about 800 to 900 nanometers in size. … Water diffuses [into the particles] at an extremely slow rate, and I think that helps protect the siRNAs fairly well.

The other nice thing about the particles is that they can be freeze dried, which is very important for collaborations because we can give the particles to people [as] … a solid powder and they just have to re-suspend it and do the animal work.

How did you test the nanoparticles in vivo?

This is all geared toward treating inflammatory bowel disease or colitis. The interesting thing about inflammatory bowel disease is that it is completely driven by a protein called TNF-alpha, which is the culprit in many inflammatory diseases. There are actually very good TNF-alpha blockers out there, but the problem is that TNF-alpha is also needed for normal immune function. If you systemically knock out TNF-alpha, you get various types of pathologies, in particular cancer, immune dysfunction, and infections.

So it would be really nice if there were a way to selectively knock out TNF-alpha, and that was why we chose it [as a target] for the siRNA. The other important thing about TNF-alpha is that its major source is macrophages, and macrophages are going to be pretty much the only type of cell that can take up a one-micron-sized particle.

So if we put TNF-alpha siRNAs in these polythioketal particles and if we deliver them orally, a lot of things happen to our benefit. The first thing is that oral delivery is tremendously convenient, and the other is that there is natural targeting to the diseased area because it's very unlikely our particles are going to go from the intestine or colon or stomach because they are just too big to diffuse into the blood and inhibit TNF-alpha in other regions.

We were anticipating that we were going to get a localized effect of TNF-alpha inhibition in the GI tract, and, just because of the chemistry of the particles, it would be localized to only the area of disease.

The key experiment was that we put TNF-alpha siRNAs in the particles, orally gavaged [them] to mice, and induced inflammatory bowel disease. We showed by RT-PCR that we can suppress TNF-alpha production by about a factor of 40, very close to baseline levels, and get a significant therapeutic response in terms of clinical scoring, weight loss, and the tissue architecture of the colon, [which] stayed very similar to the control.

Did you look at siRNA localization to confirm the molecules went where you wanted them to?

We did an experiment where we fluorescently tagged the siRNAs, put them in the particles, and could show a six-fold enhancement in siRNAs delivered to the inflamed colon [as compared] to normal tissue.

What's the next step?

The big challenge with these materials now is making sure they degrade on a time scale appropriate for human use. If you make a particle that is stable to all the different environments of the GI tract and degrades when it is supposed to, that looks fairly promising for oral delivery of siRNAs.

The big question now is how long these particles reside in tissue and what can be done to accelerate their release. I see that as being the next challenge for us.

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