A Patient Man

By Eric Sauter

Over the past two years, Enrique Saez, an assistant professor in chemical physiology on the La Jolla, California campus of The Scripps Research Institute, has won two major scientific awards: a EUREKA (for Exceptional, Unconventional Research Enabling Knowledge Acceleration) grant from the National Institutes of Health and a Career Development Award from the American Diabetes Association.

The prestigious EUREKA grant funds "exceptionally innovative research on novel hypotheses or difficult problems, solutions to which would have an extremely high impact in biomedical or biobehavioral research." The grant is designed to help investigators test unconventional hypotheses or tackle major technical challenges that might not otherwise receive funding.

Saez's EUREKA project, which was funded in August, aims to produce an innovative RNAi screening platform for mammalian cells, and make the economical technology accessible to the average research laboratory. RNAi or interference RNA targets gene expression and can be used to stifle genes' effects. This technology has great potential to enable researchers to explore the human genome and discover genes associated with disease.

However, the cost of using RNAi technology today is too high for the average lab, a problem that Saez wants to solve by miniaturizing the process.

"We can do these things because we have access to resources such as the facilities at Scripps Florida," he said, "but even for us the cost is limiting. Typically, one can spend $5,000 to $10,000 on a single RNAi screen. Another limitation is that you need robotics to use RNAi to investigate the whole genome – and there aren't that many places in the US with these robotic systems."

According to Saez, the cost of the technology also affects the quality of the results. "When the cost of screening is that high, you can't run as many replications of each experiment and that has an impact on the quality of data. By downscaling the technology, researchers could run screens in duplicate, triplicate, so that when one identifies a hit, you know it's a hit, and you don't waste time chasing false positives."

So far, Saez's group has been able to make prototypes of the basic screening platform rather quickly by using standard procedures taken from semi conductor applications. The biggest challenge has been identifying the optimal surface modifications needed to allow attachment of nucleic acids and growth of cells, and efficient release and uptake of the nucleic acids by the cells at the desired time.

"The EUREKA award is for high-risk projects," he said. "Our chances of success are reasonable – they aren't zero – because we've already achieved some important technical steps, but we still have a long way to go. This new grant will give us a shot at developing a viable platform. I have great confidence and great doubts. I know that others have tried and failed at creating these miniature platforms. Fortunately, the right person came along to work in my laboratory and take on this project, Tilak Jain. Tilak is a creative bioengineer, eager to work on challenging ideas like this one. I got lucky in finding him."

Toward a Deeper Understanding of Metabolism

Saez's second major project grant, a five-year Career Development Award from the American Diabetes Association, began last year. These awards support investigators who are poised to make significant contributions to diabetes research.

This grant will give Saez a chance to expand his breakthrough work in metabolic disorders, focusing on the Liver X Receptors (LXRs), transcription factors that act as sensors of dietary components to orchestrate the body's response to nutrients such as oxysterols (short-lived derivatives of cholesterol) and control gene expression linked to cholesterol and fat metabolism.

Saez's group recently found that LXR activity could be regulated, not just by oxysterols, but also by glucose. Because LXRs act as the body's sensor of a buildup of pathogenic cholesterol, their ability to respond to both glucose and oxysterols suggests that they may be a link between hyperglycemia and atherosclerosis.

"Our studies are aimed at understanding why diabetics are more prone to develop atherosclerosis," he said. "More than 70 percent of diabetic patient deaths are due to cardiovascular disease. If we understand how LXR activity is regulated by glucose and pathogenic forms of cholesterol, we may be able to produce therapeutics that can slow development of cardiovascular disease in these patients." This work is also supported by the McDonald's Center for Type 2 Diabetes and Obesity at Scripps Research.

His findings with LXRs may also help another patient group – stroke victims. In a study published last year, Saez showed how activation of LXRs within a few hours of an incident could effectively block any ischemia-induced brain damage in animal models by reducing levels of inflammation and promoting the expression of VEGF, a growth factor that stimulates new blood vessel growth.

Saez thinks in terms of global systems and deciphering interactions and how it all works together. He keeps at his investigations, year after year, so that one day he might gain an understanding of how metabolism works.

"In something like metabolic disease, there are multiple organs involved, such as pancreas, liver, muscle, fat, and brain, and they're in constant communication," he said. "So there are all these different and highly complex interactions between tissues to consider – there's a high learning curve when you come into the lab. Figuring it all out, like figuring out the right miniaturization technology, may be possible in time. That's why we keep working to establish a track record of accomplishment."

The Long View

Saez's showed a propensity for hard work early on.

Saez, who is now an American citizen, was born in Madrid, lived in Venezuela for several years as a child, but spent most of his life in the United States. When he was a teenager, he went to the Hun School in Princeton (NJ), planning to stay only a year to learn English. He stayed on a scholarship and in his senior year was already taking courses in molecular biology and neuroscience at nearby Princeton University and volunteering in a chemistry lab, where he acquired a taste for biomedical research.

Princeton offered him a scholarship and that taste for research came in handy.

"I was lucky and successful early on," he said. "The first few projects I did in the lab worked right away. Then I went off to graduate school and suddenly realized there are many walls that you hit in science."

He hit a couple of those walls during his graduate work at Harvard in genetics – focusing on the role of the c-Fos oncogene in cancer development – but, more importantly, he did his work at a time (1989 to 1995) when the merits of sequencing the human genome were the subject of spirited debate. As the Human Genome Project progressed Saez saw the power of technology to revolutionize the course of research.

"There were people who saw the potential this wealth of information would bring to science," he said. "But to be honest, at the end of the day, it was the technology developments that made the effort feasible. Without them, we would not be where we are today."

Saez pursued his technology bent at the Salk Institute, where part of his postdoctoral work focused on developing the edcysone gene switch, a way to turn on and off genes at will in cells and animals, a property that is useful not just for basic research, but also to enhance the safety of gene therapy.

Today, he is still passionate about the use of technology in pursuit of biological questions. "We want to develop technology to advance biology."

Admittedly, both his current projects, building a miniature RNAi screening platform and seeking a deeper understanding of the body's metabolism, may take awhile.

But no matter. Saez has the optimism of a patient man. One day both projects will succeed and, although it might be a slightly longer stretch, the two research pathways may, in fact, merge or at least intersect.

"Sometimes you just need patience," he said, "and I'm a patient man to begin with. I know what we can contribute to our understanding of metabolic disease with the diabetes grant but these experiments may take years to yield a conclusive answer, so one has to work hard and hope for the best. Of course, I'd like to know the answer much sooner, but there are limitations and I want to know the real answer."

Send comments to: mikaono[at]scripps.edu

Assistant Professor Enrique Saez is optimistic that in time his work will lead to a better understanding the body's metabolism and that he will be able to develop a broadly accessible RNAi screening technology. (Photo by Kevin Fung.)