By Mike May

Because proteins perform an overwhelming array of essential structural and catalytic roles, alterations in the structure or expression pattern of individual proteins frequently constitute the etiology of disease. The presence of one or more specific proteins, such as mutant forms of the tumor suppressor p53, can cause or enhance the progression of many cancers. Overexpressed mutant—or otherwise defective—proteins can also contribute to heart disease and other medical conditions. “There are a huge number of diseases where we know what protein to take out,” says John Hastewell, head of the biologics center of the Novartis Institutes for BioMedical Research, Cambridge, MA, “but we haven't been able to touch lots of these proteins with antibodies or small molecules.” That just might change through therapies based on RNA interference or RNAi.

The initial steps in RNAi depend on the starting material. For instance, long, double-stranded RNA (dsRNA) molecules can be introduced from outside the cell. Once in the cytosol, a ribonuclease III-type protein, called Dicer, cleaves the dsRNA into 21–25 base pair duplexes, termed small interfering RNA (siRNA). The siRNA can come from several sources: (i) it can be processed from Dicer, as described; (ii) siRNA can be delivered directly to the cell; or (iii) DNA-based vector systems can create short hairpin RNA (shRNA) that the cell processes into siRNA. In any of these cases, the siRNA will become incorporated into a multiprotein complex called RNA-induced silencing complex (RISC), which contains the protein argonaute, a catalytically active RNase. This multi-enzyme complex unwinds the siRNA, releasing the sense strand, but keeping the antisense strand bound to the complex. The antisense strand of the siRNA can then hybridize to a complementary strand of messenger RNA (mRNA). Such a binding event turns on the nuclease activity of argonaute in RISC, cleaving the target mRNA. The damaged mRNA is then degraded, making it possible for RNAi to significantly reduce the expression of a specific gene.

Unraveling the scientific basis of RNAi earned Andrew Fire of Stanford University, Palo Alto, CA, and Craig Mello of the University of Massachusetts Medical School, Worcester, MA, the 2006 Nobel Prize in Physiology or Medicine. The question now is: can RNAi move from the research lab to the clinic?

Overall, guarded optimism pervades this area of translational research. Clinical trials are already underway at three companies: Acuity Pharmaceuticals in Philadelphia, PA, and SIRNA Therapeutics, based in San Francisco, CA (recently acquired by Merck & Co, Whitehouse Station, NJ), are testing RNAi treatments for age-related macular degeneration (AMD), and Alnylam Pharmaceuticals in Cambridge, MA, has an RNAi product in trials for respiratory syncytial virus (RSV). Behind those candidates, even more approaches exist at the basic research stage. As Hastewell says, “RNAi gives us a chance to tap the knowledge base that we've had for many years.” Reaching that goal—using the available knowledge about which proteins to eliminate for specific diseases—demands overcoming some daunting obstacles.

Delivery of siRNA into Cells

To make this system work, the RNA must be delivered to specific cells, and the human body has formidable mechanisms in place to prevent this type of invasion. “siRNA duplexes are not normally found in the blood,” says John Petrovich, chief executive officer of Calando Pharmaceuticals in Pasadena, CA, “so enzymes find the siRNA and chew it up—rapidly.” That causes stability issues for siRNA, especially if injected into the bloodstream.

That's only the first stumbling block, though, because getting siRNA to the vicinity of the right cell does not guarantee getting it inside the right cell. “There are no receptors on the outside of cells to snag siRNA and pull it inside,” says Petrovich. So siRNAs must be chemically modified to not only survive in the circulatory system, but to be taken up by the correct cell types.

Calando accomplishes these goals by conjugating the siRNA to a polymer structurally related to cyclodextrin. This process creates nanoparticles roughly 50–100 nm in diameter consisting of polymer, targeting ligands, and siRNA. Integration into nanoparticles protects the siRNA from enzymatic destruction in the bloodstream, and the targeting ligands target it to specific tissues. Then, the entire nanoparticle is taken up into a cell through endocytosis. Upon entry, the siRNA is released from the nanoparticle and enters the silencing pathway to block its mRNA target. So far, Calando scientists have successfully targeted tumor and liver cells using this technique.