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Technique Might Be Used to Treat Multiple Diseases

The field of RNA interference has moved quickly. It took just eight years after the surprising revelation of RNA-based gene silencing in worms in 1998 for the discoverers to take home a Nobel Prize for their work. In the interim, RNAi took over among researchers as the number one tool for probing gene function. The race continues in the effort to unlock the enormous therapeutic potential of RNAi, which could yield exquisitely focused therapies for many diseases.

The main problem with RNA-based medicines turns out to be a purely practical one. The RNAs, while small, are still much larger than conventional drugs, subject to degradation in the bloodstream, and not likely to pass into cells unassisted. The challenge is to direct intact RNAs to the right tissues in the body and then usher them into cells.


The solution could lie in nanoparticles, according to new work from Motomu Shimaoka, junior investigator at the Immune Disease Institute and recently appointed HMS associate professor of Anaesthesia at Children's Hospital Boston.

In a paper published in the Feb. 1 Science, Shimaoka and colleagues use a novel nanoscale delivery vehicle to target therapeutic RNAs to immune cells, effectively quieting intestinal inflammation in a mouse model of Crohn's disease.

"The work is first of all a technological advance for siRNA therapies," said Shimaoka. However, the work also reveals some interesting biology, including a potential new target for treating inflammatory disease.

"We tackled probably the most difficult problem," Shimaoka explained, noting that inflammation presents more than the usual challenges for siRNA targeting. The immune cell targets can be almost anywhere in the body. And they are well known for their resistance to taking up RNAs. To get to the cells, Shimaoka and postdoctoral fellow Dan Peer have developed a multilayer, multifunctional nanoparticle that directs the RNAs toward their target cells, protects them during the journey, and helps them enter the cells once they reach their destination.

Peer, the lead author on the new report, worked from existing delivery methods that entrap siRNAs in bubbles of fat, which serve to float the cargo through the circulation and, at the same time, protect them from degradation. Peer, however, made several crucial adjustments to the shipping containers. First, he substituted naturally occurring fats for commonly used artificial lipids, which have been shown to be toxic. Then, he added a sugar coating, also derived from natural sources, which stabilizes the wobbly lipid vesicles into uniform spheres about 100 nm in diameter. The sugar also helped protect the vessels from being recognized and destroyed by the immune system of the recipient.

To ensure proper delivery to the target cells, Peer added an address to the outer surface of the particles. For that, he used a monoclonal antibody to the integrin beta7 cell adhesion receptor, a protein present at high levels on the surface of some immune cells. In particular, the beta7 protein is elevated on cells that localize to the lining of the intestine, causing a chronic and painful inflammation of the digestive tract in Crohn's disease and inflammatory bowel disease.

For the payload, Peer mixed negatively charged siRNA molecules with protamine, a positively charged protein that compacts the nucleic acid. Putting it all together is as simple as swelling the dehydrated nanoparticles in water in the presence of the siRNAs, which become encapsulated as the vesicles enlarge. By this process, the investigators found they could consistently pack in about 4,000 siRNA molecules per particle, or about 500 siRNAs per targeting antibody. This is far more than previous antibody targeting technologies, which max out at five to 10 siRNAs per antibody.

In mice, the particles turned out to be potent and precise couriers. Only cells bearing the beta7 integrin took up the siRNAs, as expected. When injected into the bloodstream, the nanoparticles homed to immune cells in the intestine and spleen. In mice with colitis, 35 percent of the dose ended up in the intestines. The nanoparticles turned out to be the most potent RNAi delivery technology demonstrated so far in mice, with a minimal dose sufficient to knock down expression of a test protein in the immune cells.

Putting Brakes on Inflammation

But could the technology treat disease? To test that, the researchers had to choose a target for their siRNA. They knew the cell cycle regulatory protein cyclin D1 becomes more abundant in immune cells during inflammation and that it plays a role in the expansion of the harmful cells, but no one knew if the protein played a role in disease. _Shimaoka and his group were in a good position to answer that question. Using their nanoparticles to deliver siRNA against cyclin D1 in mice suffering from experimentally induced colitis, they found they could lower cyclin D1 messenger RNA and protein and that this, indeed, reduced the proliferation of the cells.

The knockdown revealed another, unexpected activity of cyclin D1 in the immune cells. Independent of its effects on cell proliferation, cyclin D1 drove the production of harmful inflammatory proteins including TNF-alpha and other cytokines. Knocking down cyclin D1 blocked production of these inflammatory proteins, with the added benefit that it did not affect the output of beneficial, anti-inflammatory cytokines, the researchers showed.

As a result, mice treated with cyclin siRNA showed a marked improvement. They showed far less damage to their intestinal lining and fewer invading immune cells there. Untreated mice lost weight and had low hematocrits from intestinal bleeding; siRNA treatment relieved both these symptoms.

"This action of cyclins is completely new," said Shimaoka, and it could provide an opportunity for treating human inflammatory diseases. Beyond that, the new delivery technology should allow the elucidation of many more targets and facilitate drug discovery by providing researchers with critical evidence about the actions of other proteins in immune cells in vivo.

"This is really a platform technology," added Peer. "It gives us the opportunity to use a variety of antibodies to target different cell surface markers, not just integrins. Also, you can definitely use different siRNAs." With time and optimization for each system comes the possibility of treating many diseases, and the researchers report they are now focusing on treatment for other leukocyte diseases, including leukemia and HIV infection.

Courtesy Motumu Shimaoka

Molecular manufacturing. Stepwise assembly of targeted siRNA-laden nanoparticles begins with multilayered lipid vesicles (MLV), which are extruded under pressure to form unilamellar vesicles (ULV). A sugar coat of hyaluronan provides stability and a tethering site for anti-_integrin monoclonal antibodies. The completed shell is freeze-dried and rehydrated in the presence of siRNA-protein complexes to produce an _integrin-_targeted, stabilized nanoparticle (I-tsNP) entrapping a therapeutic RNA. Incorporating other antibodies or siRNAs allows the possibility of targeting other cells and diseases.

In that effort, Francis Szoka of the University of California, San Francisco, sees a reason for hope. In a commentary accompanying the research article, he writes, "Peer et al. offer a cautious optimism for siRNA becoming a therapeutic reality to treat human diseases in the coming decade."