As an 11-year-old in Sweden in 1979, Lars Brandén read about retroviruses in a popular science magazine, and it occurred to him that they could introduce healthy genes into cells. “I told my father I would do gene therapy when I grew up,” he said, laughing. “When you’re 11 years old, you don’t know. But I did.”
Now director of the Yale Center for High Throughput Cell Biology, Brandén, 41, has kept that promise, and moved on to another frontier of genetic research, in keeping with his propensity for exploration and individualism. He wears his hair long and has been known to sport a handlebar mustache. The Thor’s Hammer around his neck is a replica from the era of an ancestor who was a henchman of a 12th-century Norse king. He smokes cigars and feeds the piranhas in the tank in his office with meat on a string.
That office is on the West Campus, where Brandén and his colleague James E. Rothman, Ph.D., professor and chair of cell biology, Wallace Professor of Biomedical Sciences, and executive director of the center, run the first laboratory to move into the 1.6-million-square-foot former Bayer facility, which Yale purchased in 2007. The center is housed in a building just inside the West Haven town line, a few hundred feet from the tiny Oyster River. In the lobby is an old-fashioned popcorn wagon, a coffee table that used to be a packing crate for lab instruments, and a large photograph of 16 smiling scientists. Apart from several staff members from the Peabody Museum, these 16 people are the first Yale researchers to colonize the West Campus. There, in the first of three core science facilities planned for the West Campus, they are quietly performing genetic research on a scale that beggars the imagination.
Starting from scratch
When Brandén and the team arrived at Building B-31 on the West Campus in July 2008, they started from scratch. “Not even the toilets were working. … We had to buy pens, even. We had no phones—nothing,” he says. The rooms were then gutted and painted, data jacks were installed, and the researchers began placing orders for the most state-of-the-art equipment available: automated liquid and plate handling platforms, an Opera confocal imaging system, a 24-CPU Linux computer cluster, and many other items, some of them custom-built.
But Brandén is used to being the first man in a new environment. The Center for High Throughput Cell Biology is the third such infant facility he has joined. In 1995, he was one of the first scientists to work at the new southern campus of the Karolinska Institute, just outside Stockholm. There he kept his childhood vow by studying gene therapy while earning his doctorate in cell and molecular biology. Several years later he helped design and set up Columbia University’s Genome Center and its high-throughput chemical compound screening facility, at which he served as associate director until last summer’s move to Yale with Rothman and several other colleagues.
Brandén’s experience in New York made the setup process faster. Whereas the Columbia institute took some 18 months to get up and running, Yale’s was ready in 13, even though it is a more complex operation.
Perhaps unsurprisingly for a man who takes pride in his Viking ancestry and sometimes hunts his own food with bow and arrow, Brandén relishes the role of pioneer. At a new campus, he said, one has “freedom to explore novel approaches for doing science without having a preconceived idea as to how it should be done. [It gives] you more freedom to define what direction you want to go in yourself instead of having to battle old fiefdoms.”
First teams to use the core
Daniel DiMaio, M.D., Ph.D., vice chair and Waldemar Von Zedtwitz Professor of Genetics, and his team are among the first Yale scientists to make use of the center’s services. Brandén’s group helps researchers learn how cellular processes unfold by examining the function of every gene in the human genome more or less at once. In each of thousands of tiny adjacent wells, a different gene is deactivated to determine how cells react. Laser microscopes scan the wells and send data to computers with between 10 and 80 terabytes of disc space. The final result is an inventory of genes that may be important to the cellular process being studied as well as information about the pathways those genes may affect, loaded on a jump drive that fits on a researcher’s key ring.
DiMaio and graduate student Alex Lipovsky study the human papilloma virus (HPV), 13 of whose 130 types can cause genital warts and cervical cancer. The researchers are interested in the crucial first steps of infection: how the virus enters human cells and expresses its genes. All HPV types consist of a protein shell around a length of DNA that encodes between six and eight genes. Human cells and the virus’ protein coat interact in such a way that the cells allow the virus to enter. The center is working with DiMaio’s team to design an assay to pinpoint the cellular genes involved in this event. Lipovsky began by creating a pseudovirus whose protein coat is identical to HPV’s but whose innards contain only a gene encoding a green fluorescent protein. This stand-in for the real virus is later mixed with cells in which one gene has been selectively deactivated.
The experiment takes place in tiny volumes and huge numbers. Within the center’s labs are machines that hold 70 plates, each plate containing 384 wells a few millimeters in diameter and holding 50 microliters each. Robots inject 1,000 human cells into each well. Then “bullets” that knock out specific genes are introduced: small interfering ribonucleic acids, or siRNAs. These short lengths of RNA, discovered 10 years ago in plants, are now available in synthetic form, several for each gene in the human genome. The siRNAs effectively disable the gene in question—a different gene in each well—and are available to researchers in vast libraries. The siRNAs are mixed with the cancer cells for 48 hours; then the pseudovirus is introduced.
Cells that take up the pseudovirus glow green. Some cells take it up less effectively than others, and some never take it up at all. A cell that doesn’t glow either did not take up the virus, prevented viral transit to the nucleus, or failed to express the green fluorescent protein or express the virus—and that is important, since it may mean the specific gene silenced in that well by that siRNA is critical to virus uptake or gene expression. Those variations in the brightness of the green glow—the range of fluorescence—are examined, quantified, and sorted out by the automated microscope and software, leading to hits that may be crucial genes in the process of HPV infection. DiMaio’s group of researchers will then go on to study the candidate genes in their own lab.
Though suppressing genes to see how that alters cell behavior sounds straightforward, Lipovsky said that “the devil is always in the details.” There are countless technical steps that must be refined to perfection before the assay can be run, and it takes months of planning to hash them out. How long and at what temperature should the cells be incubated? How should the growth medium’s greater viscosity at the edges of the plates be accounted for? Many commercially available siRNAs have never been tested—will they do their job and knock out the genes? The center takes pride in its ability to guide researchers through these complex questions of experimental design. For several months Lipovsky spent several days a week driving to West Campus and meeting with assay-development specialist Michael Wyler, M.A., also formerly of Columbia, to discuss these details.
This kind of collaboration with clients, Brandén says, sets the Yale center—which charges both Yale investigators and outside parties a fee for service to cover some of its costs—apart from many of its competitors. At another university that offers high-throughput siRNA screening, he said, “referring investigators have to come and do the screens themselves. So you have access to instrumentations, and access to the facilities, but basically, you’re also inviting everyone to do the same mistake over and over again. ... We have a different approach, we’re taking the investigator here, we work with him to help him develop their assay, then we run it for him.” Wyler added that the approach at Yale is both fun and science-oriented. “We actually interact with the PIs, sit down, have science conversations, plan out the assays correctly, and work with them from the beginning to the end.”
“You can definitely feel the enthusiasm. With this screen, we’re getting an integrated picture of how [HPV] infects the cell,” said Lipovsky. “And this has never been done before.”
A revolution in high-throughput screening
High-throughput screening (HTS) technology, which can also employ chemical compounds rather than siRNAs to knock out genes, was developed in the late 1980s and has since been a mainstay of drug discovery within the pharmaceutical industry. Only in the last 10 years has the technique moved to academia on an industrial scale. Yale is joining the wave of academic institutions to institute HTS capacity; other pioneering groups include the Broad Institute of the Massachusetts Institute of Technology and Harvard, and Stanford’s High-Throughput Bioscience Center.
Relying as it does on complex and costly robots, laser microscopes, and vast computing power, HTS might be thought of as research on a scale appropriate to the spectacularly complicated workings of the cell. A cell’s machinery comprises a series of chemical signals that, like a microscopic Rube Goldberg contraption, each set off a different process that cascades toward an end result. Unlike a Rube Goldberg gadget, though, these signals proceed across intricate networks rather than linearly.
Teasing out how each gene product interacts with the next one has historically taken years; to date researchers have pieced together only small parts of human cell networks. Most previous methods relied on chemical compounds alone to knock out one part of a cellular process. But the chemicals can deactivate more than just the process in question, complicating the analysis. “All drugs like that are dirty ... which made it very cumbersome to delineate,” said Brandén. The tidy specificity of siRNAs—their ability to deactivate a single gene—is part of what has allowed the HTS revolution to take place. Also, advances in automation and computing now allow thousands of experiments to occur within a day. The rich lodes of data from an HTS assay can be examined by advanced software for hits, or promising results. Many of the hits lead nowhere, but a few will point the way to genes or other compounds that are important to the process being studied. Gradually, a network of intracellular interactions becomes clear and can be mapped out graphically on powerful computing platforms. And once those networks are understood, scientists can begin to design drugs for tweaking them.
The frontiers so far seem limitless. Once one cell type’s networks are understood, the researchers can explore those of related cell types to pinpoint important differences. Genetic variations will lend yet another dimension to the hunt. “If you understand the differences,” said Brandén, “then you can also understand the side effects of drugs, how diseases are progressing in different tissues, and why they behave as they do.”
With dozens of CPUs crunching the data—and the center is able to tap up to 1,000 more at Yale’s High Performance Computing cluster in downtown New Haven—the sheer scale of the work is breathtaking. But the size of the human genome makes it necessary. After a half-century of research, only about one-tenth of the human genome’s function is understood. Some 20,000 to 25,000 genes are thought to exist, but how they and their protein products interact is still largely a mystery. High-throughput screening speeds up discovery by orders of magnitude. Currently, the center has the instruments to run experiments on 200,000 wells per day, but that level of production would need a team many times the size of the present.
As part of the center’s long-term plans, Brandén expects to design several hundred such cell-based assays that will allow an exploration of the entire human proteome. “If you cover the whole proteome, you can then get supremely detailed information about any unknown gene. You can profile drugs to understand what they would do on a global level.” This approach would vastly reduce the number of animal subjects needed in trials, he said. “You can actually understand what’s happening before you go there. You can direct the efforts of medicinal chemistry.”
The center will try to break even and it is run as a business. A small, early investment allowed Brandén to hire a marketing company and contact cell biology investigators in academic and industry centers around the world, thus creating a targeted database of potential customers.
The Center for High Throughput Cell Biology is a model for two other core research facilities planned for West Campus. A second facility will provide gene-sequencing services, and will be led by Richard Lifton, M.D., Ph.D., chair and Sterling Professor of Genetics. The third group, which will perform high-throughput screens of drug-candidate chemical compounds, will be organized and overseen by Craig Crews, Ph.D., professor of molecular, cellular, and developmental biology. Thanks to the campus’ vast size and resources, the three cores, expected to be up and running by December, will boast an information-gathering capacity rare in academia.
And the cores in turn will support departments and programs across the university, including five new interdisciplinary research institutes: the Institute of Cell Biology, also led by Rothman; and institutes of chemical biology, cancer biology, microbial diversity, and systems biology. Like the Center for High Throughput Cell Biology, these institutes will comprise groups of researchers from across the sciences, including engineers, chemists, geologists, and forestry experts as well as biologists of every stripe. “The strong sense of the science community at Yale and elsewhere is that it is going to be important to mix up these disciplines a little bit. There will always continue to be really strong science coming out of the core disciplines, but the most exciting opportunities might lie at the intersections [of different disciplines],” said Michael J. Donoghue, Ph.D., vice president for West Campus planning and program development. “One of the ways that the West Campus can be useful is to break down those barriers by actually co-locating people from those different disciplines. It provides an opportunity to do something you can’t really physically do on the central campus.”
The wild, wild West Campus
In keeping with that wild West Campus culture of diversity, the center’s team members have a broad spectrum of training, backgrounds, and interests. (They also hail from several different countries; their potlucks, featuring international cuisine, are reportedly superlative.) Bioinformatics investigator Phil H. Williams, Ph.D., grew up in Horseshoe Bend, Ark., where he worked as a volunteer firefighter and helped his father run a lawnmower-repair business. After his father bought an IBM 386 to keep track of inventory, Williams was hooked, developing a deep fascination with computers. He eventually left Horseshoe Bend and studied bioinformatics at the University of Arkansas. Research informatics scientist Marie-Aude Guié, M.S., is a computer scientist who hails originally from the Côte d’Ivoire. She puts up with good-natured teasing about being a princess, as her father is king of their ethnic group, the Baoulé, in her home country. Ashima Bhan, Ph.D., is the team’s cell culture biologist and has a background in toxicology. Informatics director Adrian Poffenberger, M.S., who grows carnivorous plants in his office, has studied biology, mathematics, and cheminformatics, and has 14 years’ experience in drug discovery research.
With researchers of such disparate backgrounds bringing their ideas to the table, “intense brainstorms” are the norm, and Brandén says team meetings are a lot of fun. “We’ve had more positive science interactions in the last six months here than we had over four years at Columbia,” he said. (The transition from New York to suburbia also appears to have gone relatively smoothly. Brandén has a house in the Woodbridge countryside where he barbecues on his riverside deck, while Wyler, formerly a Brooklynite, says he likes being able to drive to work and park in front of the building.)
The center’s HTS assays hold the potential to reveal more information about cells and in greater detail than has ever been possible at Yale. HTS, said Lipovsky, is itself a marker of a shift toward greater complexity in science. “If we’re to make the next leaps in science, we need to understand not only how genes work by themselves, but also how they work together,” Lipovsky said. “We need to understand processes, not just genes.” He is excited not only for the HPV results that will come from his carefully designed assay, but also to be a part of the center’s maiden voyage. After all, he said, “the reason I’m in science is to pioneer things.”
For his part, Brandén says he is in the perfect job. “I’m dying to know things,” he said. “I’m a very curious kind of person.” With high-throughput screening, “you’re exposed to new science every single project that you run. And the allure of visualizing and understanding a complete transduction network—not only one pathway, but the global one—that is intense.” YM