Smart Cells as Disease Sensors

One could call the field of synthetic biology precocious; for its young age it's unusually advanced, and it seems to be maturing with every new development, research paper, and application. As the field has moved from protein engineering to whole pathway design to constructing entire synthetic organisms, synthetic biology has come a long way since MIT researchers first started batting around the parts a decade ago. However, the focus is still on how best to apply the principles of engineering design to tweaking, as well as creating entirely new, biological systems.

Using gene circuits to elucidate function is nothing new; using these circuits to build molecules or organisms for improved biotech or biomedical applications is where most of the cutting-edge research is being done these days. One such application of some of this research can be seen in using circuits to design intelligent therapeutic molecules and cellular systems.

At her Caltech lab, Christina Smolke is engineering molecules and cells to better recognize and treat disease. "So what we'd like to have is a more information-rich therapeutic molecule, and the ultimate goal would be to have something that has targeting capability so that you minimize undesired side effects," she says. A so-called smart molecule, she adds, could get inside a cell and assess whether that cell is diseased or healthy, and then self-activate to either deliver a therapy or effect some other change in the cell.

That could go a long way toward establishing the particular phase of disease for a patient, based on biomarkers representative of disease state. "We want our therapeutic molecules to be able to detect those altered levels," Smolke says, "[and] then they will self-activate and particularly alter levels of proteins in the cell that will reprogram cell function." For instance, in cancer these molecules would promote apoptosis; in a metabolic disease, they would alter enzyme levels back to a normal state.

Smolke is also working on a project to engineer whole cells. In collaboration with the City of Hope's Mike Jensen, she is working to create modified T cells that will specifically respond to particular disease biomarkers. "You're essentially just making modifications to the patient's T cells by putting in synthetic DNA," she says. "In this case, [you] use the same sort of information processing and regulatory molecules but program different input/output functions into them. You put them into your immune cells so that now the immune cells are programmed to target the disease cells — and also to specifically only be activated when the molecular markers of that disease are being received by the immune cell."

Both projects are still in the cell culture phase, but a larger end goal is to go beyond building just one molecule or one cell to creating a framework that others can use to build any type of cell for various diseases. Building more reliable parts and determining the standards by which they can all fit together are key to advancing the field, Smolke says. "The question is, can we build a framework in which we can assemble many, many such molecules where the specifics of the input and output functions and the information processing function of the molecule differ, but the framework is the same?"

A better bacterium

At synBERC, hosted by the University of California, Berkeley, Chris Anderson is leading the Tumor Killing Bacterium Project, which aims to modify bacteria to localize and destroy cancer cells. For three years, Anderson's lab has been trying to engineer E. coli bacteria that can be safely injected into the bloodstream and then localized to invade and kill cancer cells. "Synthetic biology offers a lot in terms of how you might approach engineering any biological agent to be a treatment for cancer — be that bacteria, viruses, T cells, whatever," he says.

Anderson uses a nonpathogenic lab strain of E. coli, manipulating its genetic code to create a smart microbe that can sense its environment and then react. "They're always sensing the environment around them," he says, "and what they're looking for are specific cues that would tell them that they were in the tumor microenvironment." Input information comes in the form of cues like variable concentrations of oxygen, glucose, or cholesterol, or the presence of specific sugars on the surface of cancer cells, for instance. Following this input, the cells kick in and start "performing logical operations in the cell to make a decision as to whether they think they're in a tumor or not," Anderson says. If they are, they will activate to invade the tumor cells around them.

Anderson's work wrestles with the challenges of actually controlling bacterial growth within the body as well as the immune response to the bacteria, but overall, his team's technique serves as a test ground for building a large synthetic system — a whole organism — as much as it does for finding a novel cancer therapy.

"I think realistically our end goal is to take this to a point where either the idea has become popular enough in science that there are lots of people doing it, or we get to the point where the level of development is such that the experiments that really need to be done aren't in vitro, they are animal or human trials," Anderson says. "We're not that far along that we could actually treat an animal with cancer and have something happen. But we are at the point where we should be able to control the immunological properties of the bacteria and ... confirm that that works in a real animal."

Assembling parts and devices into working systems that respond predictably and robustly to changing environments is one of the larger challenges to the field. SynBERC Director Jay Keasling, whose collaborative work has engineered E. coli to become a synthetic source of the anti-malarial drug artemisinin, echoes Smolke's ideas of the need to improve both the foundation and design techniques in the field. "The whole concept behind SynBERC is to help develop this new field of synthetic biology," he says. "Can we understand how to put basic components together that can be re-used over and over again for many different applications? How do we put those components together? What are the things we need to know?"

Smolke adds, "We need to have better parts. We need to have better control over the parts that we make ... but also we need to think about what the appropriate compositional frameworks are that allow us to integrate these parts." 

Building Whole Synthetic Genomes

With the latest news of the J. Craig Venter Institute's successful assembly of the longest synthetic genome, the field of synthetic biology has heated up. In January, Venter and colleagues published results in Science demonstrating that they had synthesized and assembled the 582,970-base-pair genome of the Mycoplasma genitalium bacterium. The next, and final, step of their proof-of-principle work would be to get the genome to express itself in a living cell.

"It's quite a remarkable technical feat that they've assembled a piece of synthetic DNA that's half a million bases long," says Rob Holt of the BC Cancer Agency Research Centre.

But Venter's group is not the only one working on assembling synthetic genomes. Mitsuhiro Itaya's group at Japan's Institute for Advanced Biosciences works on genome synthesis but doesn't start with oligos. The group published work in PNAS in 2005 in which they built a piece of DNA 7.7 megabases long by cloning the entire genome of the photosynthetic bacterium Synechocystis PCC6803 into the genome of the bacterium Bacillus subtilis 168. "A subtle difference is that the Itaya group was starting with cloned fragments," Holt says. "That's probably the most important claim that Ham Smith's group can make at this point. They have started with synthetic DNA and made a bacterial chromosome that's half a million bases long."

What the Venter group is aiming for is proof of principle, and Holt thinks their previous work — published in 2003 showing that they could change one bacterial species, Mycoplasma capricolum, into another, Mycoplasma mycoides Large Colony, by replacing one organism's genome with the other's — is more interesting. "That's an important milestone, probably more important than just having built a Mycoplasma genitalium genome because it shows you can take naked DNA and get it to propagate and support an organism," Holt says. While Venter's team hasn't yet activated the M. genitalium genome, Holt suspects that it's feasible. However, because the scientists are using Mycoplasma bacteria, which lack a cell wall, this particular work won't have much practical application beyond proof of principle. Most microorganisms have cell walls; so transferring an entire genome into, for instance, an E. coli or yeast cell is very different.

"What we're trying to do that's different is to build a new genome in a host cell, so that you get a hybrid cell that has both the synthetic genome and the original host genome," Holt says. In principle, one could remove the original genome and be left with an intact cell that's being driven by the synthetic one. "So you'd get to the same endpoint, but because you're building the genome piece by piece in the host cell rather than transferring it all at once you can get around the cell wall problem," he adds. "It has more practical application because then you can engineer genomes for all sorts of industrial, important microbes."

The largest issues Holt sees for synthetic genomics are that biological systems are infinitely complex — and predicting how they'll function is challenging, to say the least. Also, synthetic circuitry is still beholden to the complexity of the host cell, which is typically E. coli. "I think that the key challenge in synthetic biology at this point is to show that there ultimately will be some utility to this," Holt says. "The whole field, we're really just at the very beginning. We're basically infants that are just learning to crawl, and can discover new things around the house that were previously out of reach."