Science Editor

A report in the April 30, 2009, issue of Nature combined a new delivery technology with a new target to debut what could help further the development of a new class of oral anti-inflammatory drugs down the road.

In their paper, the authors first described using a yeast-derived oligosaccharide to make a delivery shell, which they call GeRP. That shell, in turn, is filled with short interfering RNA that targets a kinase that appears to control production of the inflammatory cytokines via a previously unrecognized pathway.

Using their combination, the authors, who are from the University of Massachusetts Medical School, were able to block the production of TNF-alpha and interleukin-1b by macrophages in response to inflammatory stimuli.

The siRNA targets a kinase known as Map4k4, that was first described in 2006. Senior author Michael Czech told BioWorld Today that where Map4k4 sits in the inflammatory signaling cascade, and what its substrates are, is "still unclear." But its yeast homolog, STE-20, "sits at the very top of the feeding chain" of map kinases, meaning it is one of the first kinases that is activated in response to incoming signals, and phosphorylates other kinases to set off a pro-inflammatory signaling cascade that ends with the production of TNF-alpha and interleukin-1b.

Czech said the pathway is "analogous" to JNK and NF-kappa-B pathways, and could provide a new way to regulate the cytokines.

TNF-alpha blockers are used in the treatment of rheumatoid arthritis, Crohn's disease, psoriasis and ankylosing spondylitis.

While Map4k4's exact place in inflammation remains to be worked out, Czech said, what is clear is that by silencing the kinase via siRNA, "we get a big attenuation of the macrophage's response" to inflammatory stimuli activating the Toll-like receptors.

Czech and his colleagues were able to deliver the siRNA orally by making a shell out of beta-1,3-D-glucan, an oligosaccharide that is part of the yeast cell wall. The resulting shell, Czech said, is both porous and relatively rigid. But "the key feature is that it serves as a receptor ligand" for the dectin-1 receptor. That receptor, in turn, is prominent on two types of immune system cells that are found, among other places, in the gastrointestinal tract: dendritic cells and macrophages. That means the shell can be filled with siRNA and given orally; the shell will travel to the gut, be taken up by macrophages in the gut-associated lymphatic tissue, and the siRNA is released within the macrophage.

In their paper, Czech and his colleagues first used their shell to deliver anti-TNF-alpha siRNA to macrophages in the gut-associated lymphatic tissue, and found that the shells were able to stop the production of TNF-alpha. They then went on to search for possible upstream targets that might be able to prevent the production of TNF-alpha, and identified Map4k4 as a candidate target.

The authors treated cultured macrophages with glucan-encapsulated anti-map4k4 siRNA, and found that when they challenged such cells with lipopolysaccharide or LPS - a molecule that is present on some bacteria and stimulates cytokine production via its activation of Toll-like receptors - mRNA levels of TNF-alpha decreased by 40 percent in cells that were incubated with the siRNA. The same held true in in vivo experiments. When mice were given the siRNA-containing capsules orally, the siRNA was taken up by macrophages in the gut, and map4k4 expression was reduced by roughly two-thirds. The authors also found some macrophages with knocked-down map4k4 expression in the spleen, liver and lung. When the authors administered the siRNA along with a normally lethal dose of LPS to mice, the survival rate increased from 10 percent in controls to 40 percent in the treated animals.

The effect was seen with delivery of 20 mcg of siRNA per kilogram of body weight, which is one to two orders of magnitude lower than has been described in previous studies that used intravenous delivery. In their paper, the authors wrote that this high potency is "probably due to protection of siRNA against nuclease degradation . . . within GeRPs, low nonspecific binding of GeRPs en route to the gut, and high efficiency of GeRP uptake by phagocytic cells," and added that "furthermore, the siRNA loading capacity within 1,3-D-glucan shells is far greater than we used here, and can potentially deliver combinations of siRNA, DNA, proteins and small molecules."

The University has licensed the GeRP technology to Worcester, Mass-based biotech company RXi Pharmaceuticals Corp. RXi was co-founded by Czech and 2006 Nobel Laureate Craig Mello, who is also at the University of Massachusetts.

RXi, Czech, and Nature paper co-author Gary Ostroff received a three-year, $750,000 cooperative research grant to investigate the development of a range of orally delivered RNAi therapeutics using GeRPs in December 2008.