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UMMS Team Publishes In Vitro, In Vivo Data on Oral siRNA-Delivery Tech

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Researchers from the University of Massachusetts Medical School this week published a paper in Nature showing how a novel, oral siRNA-delivery technology can be used to suppress inflammation-associated gene expression in mouse macrophages in vitro and in vivo.

Dubbed glucan-encapsulated siRNA particles, or GERPs, the technology comprises hollow, porous, micrometer-sized shells that are loaded with an siRNA payload. Once delivered orally, the GERPs are designed to be taken up by M cells, which are specialized cells in the small intestine that take up antigens and carry them through the intestinal wall so they can be phagocytosed by macrophages.

"On phagocytosis of GERPs by macrophages, the acidic pH in phagosomes promotes siRNA release through the porous GERP wall," the UMMS team wrote in Nature.

Given that macrophages are "attractive targets for RNA interference therapy because they promote pathogenic inflammatory responses in diseases such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease, and diabetes," the investigators expect the technology may be useful in treating disease-associated inflammation, they noted.

RXi Pharmaceuticals holds the exclusive rights to the GERP technology (see RNAi News, 10/16/2008), which is expected to form the basis for the company's lead drug-development program in inflammatory disease (see RNAi News, 3/26/2009).

According to their Nature paper, UMMS' Michael Czech, who is also an RXi co-founder, and colleagues established that GERPs containing two siRNAs against tumor necrosis factor-alpha were extensively phagocytosed by primary macrophages, triggering target gene silencing.

"We then screened in peritoneal exudates cell macrophages for candidate intracellular signaling proteins that might control TNF-alpha expression," they wrote. "One of these was Map4k4, a germinal center protein kinase that facilitates TNF-alpha signaling itself."

The team then incubated peritoneal exudates cell macrophages with GERPs containing either Map4k4 or scrambled siRNAs. The cells were then treated with either saline or lipopolysaccharide, which activates monocytes and macrophages to produce inflammatory cytokines such as TNF-alpha and interleukin-1 beta.

TNF-alpha mRNA levels decreased by 40 percent in saline-treated cells receiving siRNA-loaded GERPs compared with those receiving the control siRNA. Meanwhile, Map4k4 silencing inhibited lipopolysaccharide-induced TNF expression by 50 percent or more, the researchers wrote.

"Map4k4 silencing in [peritoneal exudates cell macrophages] also resulted in an average 30 percent decrease in [lipopolysaccharide-induced] TNF-alpha protein secreted into the medium," they added.

5' RACE analysis confirmed that depletion of Map4k4 mRNA in the GERP-treated macrophages was due to RNAi.

To evaluate the in vivo potential of orally administered GERPs, the investigators administered GERPs containing either Map4k4 or scrambled siRNAs, conjugated with a fluorescent label, via oral gavage to mice once a day for eight consecutive days.

The GERPs were found to be taken up by macrophages of the gut-associated lymphatic tissue, which then migrated out into peripheral tissues, according to the paper. "Notably, Map4k4 mRNA expression was inhibited by 70 percent in [peritoneal exudates cell macrophages] isolated from mice orally gavaged with the Map4k4 siRNA-containing GERPs compared to scrambled siRNA-containing GERPs."

Map4k4 silencing was also examined in macrophages that had migrated to other tissues, and "significant depletions" of roughly 50 percent, 80 percent, and 40 percent were seen in macrophage-enriched cells isolated from spleen, liver, and lung tissues, respectively, in mice receiving the active siRNA.

"Consistent with the lack of gene silencing in cells from skeletal muscle, GERP-containing macrophages were rare in this tissue," the team noted. "Taken together, these data indicated that macrophages in the [gut-associated lymphatic tissue] internalize orally absorbed GERPs, undergo siRNA-mediated gene silencing, and migrate into tissues throughout the body."

The investigators also found that oral delivery of GERPs loaded with Map4k4 siRNAs blocked the increase in serum TNF-alpha protein and TNF-alpha levels in peritoneal fluid after mice were injected with lipopolysaccharide.

In addition, GERPs containing Map4k4 siRNAs were able to "significantly" protect mice from lipopolysaccharide-induced lethality through the inhibition of TNF-alpha and interleukin-1 beta production in macrophages.

Specifically, 90 percent of mice receiving GERPs containing a scrambled siRNA died 4 to 8 hours after lipopolysaccharide challenge. However, 50 percent of mice receiving the Map4k4 siRNA-loaded GERPs survived for 8 hours, while 40 percent survived long term.

In light of these data, the GERP technology "defines a new strategy for oral delivery of siRNA to attenuate inflammatory responses in human disease," the UMMS researchers wrote.

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