Mark Young and Trevor Douglas, both at Montana State University, Bozeman, in conjunction with Jack Johnsons group at the Scripps Research Institute in La Jolla, Calif., spent a number of years sleuthing the structure and assembly of viruses. They focused on the well-studied Cowpea chlorotic mottle virus (CCMV). The viral coat of CCMV, like that of many viruses, is composed of identical protein subunits that self-assemble into a quasispherical shape known as an icosahedron. This geometry forms the largest volume of a given size that can be constituted from identical subunits, notes Young. The subunits are organized into five-sided and six-sided capsomeres, which are arranged to form a pattern similar to that on a soccer ball. CCMV has gated pores that open and close according to the chemistry of its surrounding environment.
Armed with an arsenal of CCMV knowledge, the researchers began to explore whether they could redesign the capsid to both incorporate an imaging agent and zoom in on new targets. In addition, they wondered, what could be packaged inside the viral capsid in place of nucleic acid? And how could the gates be triggered to make deliveries?
It turns out that the capsid, assembled without its nucleic acid and thus no longer infectious, can serve as a highly modifiable and versatile addition to the nanoengineers toolbox. Conveniently, the empty capsid even self-assembles in the test tube or in yeast cells genetically engineered to produce subunits.
Visualizing the Threat
To conquer a metastasized cancer, physicians must identify the sites of new tumors and then selectively kill the wayward cells. CCMV capsids can potentially be engineered to achieve both goals.
For example, CCMV capsids could improve detection of tiny new tumors by magnetic resonance imaging (MRI). MRI identifies the differing responses of hydrogen atoms of water to the presence of a powerful magnetic field. Prior to being scanned, the patient may receive an injection of an imaging agent, most commonly gadolinium. The agent as currently given makes areas of interest more distinct but usually cannot resolve extremely small metastases.
Over the past two years, Young, Douglas and their colleagues significantly raised contrast levels in MRI images by incorporating the gadolinium atoms into CCMV protein shells. This promotes gadoliniums interaction with water molecules. That's because the gadolinium molecules--180 of which are woven into the 28-nanometer-diameter capsid--tend to be in higher concentration at any given location. Also, unlike todays gadolinium agent, which tends to clump, the gadolinium bound to the capsid surface keeps the atoms evenly distributed and available to interact with water.
To attach gadolinium to the capsids, the scientists exchanged the agent for the usual calcium--normally, during capsid assembly, calcium binds to the protein shell at sites between the subunits. To further knit gadolinium to the capsid, the researchers genetically engineered changes on the viral genome that optimized the binding sites for gadolinium.
Now that they had an improved imaging agent, the scientists wanted to specifically light up metastases in the MRI images. To do this, the investigators placed protein-based docking molecules on the capsids. These docking sites would bind with proteins expressed on the surface of cancer cells, so the gadolinium-bound capsids would collect at tumor sites.
Again, the investigators turned to genetic engineering, making changes in the viral genome. In fact, they found that different types of docking sites could be placed on one capsid, potentially making it possible to search for several cancer types simultaneously.
In one experiment to test the technique, the researchers attached lamanin peptide 11, a docking site for lamanin-binding protein. This protein is expressed in large quantities on the surface of many types of breast cancer cells. Tested in a cell culture system, viral capsids were able to locate the cancer cells and bind to them; cancer cell location was detected by using a laboratory technique, nuclear magnetic resonance (NMR), which works on the same principles as clinically used MRI.
By combining docking sites and gadolinium onto each capsid, the investigators could cluster the capsids around tiny clumps of cancer cells and image them in experimental systems. But what about eradicating the metastasized cancer?
Killing Cancer Cells
Bereft of its nucleic acid, the viral capsid could be a handy suitcase for transporting potent anticancer compounds to tumor sites. Over the past four years, the researchers have shown that a variety of compounds can be placed inside the capsule. They showed that some therapeutic agents used to treat cancer can be encapsulated through the viral gates or, in a few cases, can actually be manufactured in situ using the capsid as a tiny reaction vessel.
That left a final puzzle: Once docked at the tumor with the drugs, how would the capsid deliver its toxic package? The viral gate with which nature has endowed CCMV is controlled by pH, which isnt a useful trigger for delivering medication to specific sites.
Again, the scientists reengineered the evolutionary solution and designed gates controlled by redox potential (the oxidation state of a local environment, which influences the tendency of a molecule to lose or gain an electron). For initial work, the scientists have used CCMV, which, as a plant virus, does not enter human cells; however, the final delivery vehicle could be a reconfigured human virus that does slip into human cells. Since cellular interiors have a higher redox potential than the bloodstream, viral capsids could be shut tight in transit but will open their redox-controlled gates once they enter targeted cancer cells. The scientists are also developing another type of gate that is triggered by a type of radiation commonly used in cancer therapy.
The team is currently exploring how the modified virus capsules work in a mouse model system and is encouraged by promising initial results. Taken together, the four capabilities of the newly engineered capsids--high-sensitivity imaging, target finding, drug transport and controlled delivery--add up to a potentially powerful, yet minimally toxic, way to fight metastasized cancer.
Anne M. Rosenthal is based in the San Francisco area.